Significance and successful construction of stable transfection of MH7A cells with lower TRAF2 expression using lentiviral vector / 安徽医科大学学报

Objective To construct a stable synovial cell line MH7A from rheumatoid arthritis(RA)patients using lentiviral vectors that interfere with the expression of tumor necrosis factor receptor associated factor 2(TRAF2),and to study the role of TNF-α-TRAF2 signaling in MH7A abnormal proliferation.Methods Based on the design principles of human TRAF2 gene sequence and shRNA sequence,three pairs of TRAF2 shRNA interference se-quences were designed and synthesized.The primers were annealed by PCR,and a linear vector was obtained by double enzyme digestion PLKO.1-puro.The linearized vector was connected to the annealed primers through Solu-tion I,and the connected products were introduced into receptive cells.The plates were coated,and positive colo-nies were selected for sequencing.Three different recombinant plasmids of PLKO.1-TRAF2-shRNA lentivirus were constructed,and lentivirus packaging plasmids was used to package logarithmic growth phase HEK 293T cells.Vi-rus solution was collected to infect MH7A cells.At the same time,puromycin was used to screen MH7A stable transgenic strains with low TRAF2 expression.CCK-8 method,Western blot,and qPCR were used to detect the proliferation function of MH7A induced by TNF-α and low expression of TRAF2,as well as downstream signal TRAF2,P65 protein expression and mRNA levels.Results PLKO.1-TRAF2-shRNA(1),PLKO.1-TRAF2-shR-NA(2),and PLKO.1-TRAF2-shRNA(3)lentivirus vector plasmids and control group lentivirus vector plasmids PLKO.1-puro were successfully constructed.The three TRAF2-shRNA lentivirus vector plasmids and control group lentivirus vector plasmids PLKO.1-puro were respectively introduced into the lentivirus packaging plasmid of HEK 293T to obtain virus solution.After infecting MH7A cells with the virus solution,they were treated with puromycin(2.00 μ G/mL)screening and obtaining MH7A stable transgenic plants after 2 days.Through qPCR and Western blot results,it was found that the expression of TRAF2 mRNA and protein in PLKO.1-TRAF2-shRNA(1)MH7A stably transfected cells was significantly reduced compared to the negative control group.The results of CCK-8 and Western blot showed that after knocking down TRAF2 in MH7A,the proliferation of MH7A cells with low TRAF2 expression induced by TNF-α and the phosphorylation level of P65 were significantly reduced.Conclusion A sta-ble transgenic strain of PLKO.1-TRAF2-shRNA(1)MH7A cells was successfully constructed to investigate the role of TNF-α-TRAF2 signal activation in mediating abnormal proliferation of RA synovial cells.

Differentiation of insulin-producing cells from human umbilical cord mesenchymal stem cells infected by MAFA-PDX1 overexpressed lentivirus / 中国组织工程研究

BACKGROUND:Transplantation of stem cell-derived islet β cells has been considered effective for the treatment of type 1 diabetes.Human umbilical cord mesenchymal stem cell is an ideal cellular source,but with a low differentiation efficiency to islet β cells. OBJECTIVE:To explore the possibility of human umbilical cord mesenchymal stem cells modified by MAFA and PDX1 to differentiate into insulin-producing cells. METHODS:MAFA-PDX1 lentivirus expression vectors were constructed.The efficiency and potentiality of human umbilical cord mesenchymal stem cells differentiated into insulin-producing cells with three methods were compared by cell morphology,RT-qPCR,and dithizone staining[protocol A:Simple lentivirus group;protocol B:Drug(nicotinamide β-mercaptoethanol)induction followed by lentivirus group;protocol C:lentivirus and drug induction group]. RESULTS AND CONCLUSION:(1)Morphological change of cells:Cell morphology was all altered after the induction of three protocols.At day 11,human umbilical cord mesenchymal stem cells induced by protocol B showed the most cell clusters among the three protocols,appearing aggregated islet-like cell clusters.(2)Islet-related gene expression detected by RT-qPCR:Horizontal comparison of the three protocols at the same induction time point showed that the expression levels of MAFA and PDX1 genes were the highest in protocol C on day 5 of induction,and those in protocol B were the highest on day 11 of induction.Human umbilical cord mesenchymal stem cells induced by protocol B had the greatest expression of GCG gene at day 5,INS and GLUT2 genes at day 11.(3)Dithizone staining to identify zinc ions:parts of the post-induced cells were stained brownish red by dithizone on day 11.The partial small island cells were stained brownish red with a darker color(positive expression)in protocol B.(4)It is concluded that the overexpression of MAFA and PDX1 can promote the differentiation of human umbilical cord mesenchymal stem cells into insulin-producing cells.The combination of MAFA-PDX1 gene modification and drug induction is superior to the single gene modification.

Overexpression of miR-378a promotes macrophage M2 polarization and inhibits M1 polarization / 中国组织工程研究

BACKGROUND:M2 macrophages have the function of reducing inflammatory factors and promoting tissue healing.Therefore,how to regulate M2 polarization of macrophages has been a hot research topic in recent years,and some miRNAs have been found to have this function. OBJECTIVE:To investigate the effects of miR-378a on the polarization of the Raw264.7 macrophage cell line. METHODS:The M1 polarization of macrophages was induced by lipopolysaccharide and interferon-γ.Interleukin-4 induced M2 polarization and the expression of endogenous miR-378a in each cell type was detected using qRT-PCR to verify whether miR-378a was involved in the polarization of macrophages.By transfection with lentivirus as the vector of overexpression of miR-378a,the stable expression of miR-378a cell lines was screened.Macrophage M1 polarization was induced synergically by lipopolysaccharide and interferon-γ.Macrophage M2 polarization was induced by interleukin-4.The levels of M1/M2 polarization-related cytokines in the supernatant of the macrophage culture medium were determined by enzyme-linked immunosorbent assay.qRT-PCR was used to detect the polarization characteristics of M1/M2-type macrophages and the mRNA expression levels of related cytokines. RESULTS AND CONCLUSION:(1)The expression level of endogenous miR-378a in Raw264.7 cells of each group increased after macrophage polarization.(2)Compared with the non-transfected group,the expressions of proinflammatory cytokine-induced nitric oxide synthase,tumor necrosis factor-α,interleukin-6 and interleukin-1β in macrophage M1 induced polarization were significantly decreased in the miR-378a transfection group(P<0.05);the levels of inducible nitric oxide synthase,tumor necrosis factor-α and interleukin-6 in cell supernatant were also significantly decreased(P<0.05).(3)Compared with the non-transfected group,the expressions of CD206,interleukin-10 and arginase-I in macrophage M2 induced polarization were significantly increased(P<0.05);the levels of CD206 and interleukin-10 in cell supernatant were also significantly increased(P<0.05)in the miR-378a transfection group.(4)It is indicated that overexpression of miR-378a promotes the M2 polarization of macrophages and inhibits the M1 polarization of macrophages.

Overexpression of circular RNA HIPK3 prevents activation of rat microglia / 安徽医科大学学报

Objective @#To investigate the relationship between circular RNA homeodomain interacting protein kinase 3 (circHIPK3) and the activation of rat microglia (RM) cells.@*Methods @#In vitro , RM cells were cultured and randomized into normal and oxygen⁃glucose deprivation/reperfusion (OGD/R) groups , and the expression level of circHIPK3 in each group was detected by RT⁃qPCR. The circHIPK3 lentiviral vector with puromycin resistance was constructed , and the overexpression (OE) group and negative control (NC) group were set up. The optimal multiplicity of infection (MOI) for RM cells was determined based on fluorescence expression , and puromycin was used to screen RM cells stably expressing circHIPK3. The cells of OE and NC groups were treated with OGD/R , and the expression levels of ionized calcium binding adaptor molecule 1 (Iba ⁃1) and eukaryotic tumor necrosis factor receptor superfamily (CD40) were detected by Western blot. The circHIPK3 translational protein potential was analyzed by the circRNAdb database , while the potential binding microRNAs on circHIPK3 were predicted by circBank and Starbase databases.@*Results @#OGD/R down⁃regulated circHIPK3 in RM cells ( P < 0. 000 1) . The sequencing results were accurate and the lentiviral vector of circHIPK3 was constructed successfully. The optimal MOI of RM cells was 80 , puromycin at a concentration of 2 μg/ml was used to screen RM cell lines stably expressing circHIPK3. RT⁃qPCR results showed that the expression level of circHIPK3 was significantly higher in the OE group compared with the NC group (P < 0. 01) . Western blot results revealed that the expression levels of Iba and CD40 in the OE group were markedly lower than those in the NC group (P < 0. 05) . Protein translation analysis showed that circHIPK3 encoded a polypeptide of 404 amino acids with two internal ribosome entry sites (IRES) and an open reading frame (ORF) . Database analysis uncovered that circHIPK3 could target eight specific miRNAs , namely hsa⁃miR⁃3529⁃5p , hsa⁃miR⁃379⁃5p , hsa⁃miR⁃506⁃3p , hsa⁃miR⁃33 , hsa⁃miR⁃450b⁃5p , hsa⁃miR551b⁃3p , hsa⁃miR⁃193 , and hsa⁃miR⁃508 ⁃3p.@*Conclusion @#The overexpression of circHIPK3 effectively suppresses OGD/R⁃induced activation of RM cells. It has the potential to encode peptides and may act as a miRNA sponge. These findings provide a foundation for further study of circHIPK3 functions.

Lentivirus vector in gene therapy of β-thalassemia / 基础医学与临床

β-thalassemia is a common monogenic inherited blood disorder caused by mutations of β-globin gene which results to synthesis obstacles or abnormal structure of β-globin.Gene therapy mediated by lentiviral vector(LVV)is divided into gene-integration strategies using LVV to add fully functional β-hemoglobin(HBB)genes to chromosomes and gene-editing strategies using LVV to deliver specific ribozymes to hematopoietic stem cells for in situ repair of HBB genes.Improving viral titration and transduction efficiency,reducing target loss and advancing clinical trials are the main targets of these two strategies.

Packaging systems for generating SARS-CoV-2 pseudoviruses: A mini review

@#The COVID-19 pandemic has caused a huge damage to global society, not only affecting health aspect but also economics. The development of effective vaccines and therapeutics has been a critical step in the fight against this pandemic, and much research has been committed to studying the causative virus, SARS-CoV-2. Pseudovirus, a valuable research tool that allows investigating SARS-CoV-2 in a safe and controlled environment, has gained significant attention in recent years. It is a promising tool for examining the behaviour of the viral envelope protein and the development of vaccines and therapeutics. Finding an optimized pseudovirus system is crucial for various scientific and medical research applications, especially in the study of viral infections, immune responses and vaccine development. It plays a crucial role in advancing our understanding of viral diseases to combat with the new variants.This minreview focuses on the concept of pseudovirus systems and factors affecting the pseudovirus yield.

Construction of a chimeric antigen receptor CAR THP －1 cell line targeting HER2 / 安徽医科大学学报

Objective@#To obtain chimeric antigen receptor macrophages ( CAR-M) targeting HER2 stably transfected．@*Methods @#CAR lentivirus vector targeting HER2 was constructed and infected with human monocytic leukemia cell line (THP-1) ．CAR THP-1 cells with green fluorescent labeling were selected by sorting flow cytometry and continued to be cultured in vitro．The CAR THP-1 cells targeting HER2 were co-cultured with the endometrial cancer cell line Ishikawa with negative and positive HER2 expression，and their targeted phagocytosis of CAR-M to HER2 positive tumor cells was detected by imaging flow cytometry ，and the targeted phagocytosis efficiency of CAR-M to HER2 positive tumor cells was detected by flow cytometry. @*Results @#CAR lentivirus infection with THP- 1 cells was less efficient ; After co-culture with cancer cells，flow cytometry and imaging flow cytometry showed that CAR THP-1 cells had enhanced phagocytosis of HER2 positive Ishikawa cells compared with the empty body group (P＜0. 01) ．@*Conclusion @#In this experiment，CAR THP-1 cell line targeting HER2 was established by constructing CAR lentivirus vector and transfecting THP-1 cells ，and it was proved that CAR THP-1 could phagocytize HER2 positive Ishikawa cells through specific targeting.

Stable expression of shRNA for the control of recombinant adenovirus replication

Preventing the replication of adenovirus could have practical uses, such as controlling infection with wild-type virus or in applications involving recombinant vectors. Mainly transient methods have been used to inhibit adenovirus replication, including siRNA or drugs. Here, we tested whether stable expression of shRNA designed to target hexon, Iva2, or pol can inhibit the replication of a recombinant adenoviral vector, Ad-LacZ (serotype 5, E1/E3 deleted), in 293T cells. Significant knockdown correlating with reduced Ad-LacZ replication was achieved only when hexon was targeted. Cell sorting and isolation of cellular clones further accentuated knockdown of the hexon transcript, reduced protein levels by more than 90%, and diminished adenovirus production. As visualized by transmission electron microscopy, the cellular clone expressing the hexon-specific shRNA yielded 89.2% fewer particles compared to the parental 293T cells. Full scale production followed by purification revealed a 90.2% reduction in Ad-LacZ biological titer. These results support the notion that stable expression of shRNA can be used as a means to control adenovirus replication.

Padronização de um ELISA indireto a partir da utilização de uma proteína de superfície do vírus da anemia infecciosa equina como antígeno para o diagnóstico dessa enfermidade / Standardization of an indirect ELISA using a surface protein of the equine infectious anemia virus as an antigen for the diagnosis of this disease

A anemia infecciosa equina é uma importante enfermidade que acomete os equídeos em todo o mundo, se apresentando de forma aguda, crônica e assintomática causando grandes prejuízos para a economia tanto para criadores que vivem do trabalho desses animais quantos aos criadores que investem no melhoramento das raças, impedindo o acesso ao mercado tanto nacional quanto internacional. O Ministério da Agricultura, Pecuária e Abastecimento considera o IDGA como teste oficial para diagnóstico dessa enfermidade, porém essa técnica é demorada e muita vez acaba sendo subjetiva, dependendo da experiencia particular de cada Laboratorista. Além de não conseguir detectar animais no início da infecção. Logo, a necessidade de se buscar novas técnicas como o ELISA indireto que aperfeiçoem o tempo de análise dos resultados, facilita a automação e obtém resultados confiáveis. O estudo realizado teve como objetivo padronizar uma técnica de ELISA indireto utilizando uma proteína de envelope viral GP90 como antígeno para diagnóstico da anemia infecciosa equina. Avaliando o desempenho do teste a partir da sensibilidade, especificidade e valores preditivos positivo e negativo. Os valores obtidos foram: 91,11%, 93,33%, 91,11% e 93,33% respectivamente. Concluiu-se que o teste apresenta bom desempenho, além da possibilidade de detectar amimais positivos no início da infecção.

Equine infectious anemia is an important disease that affects horses all over the world, presenting in an acute, chronic and asymptomatic way, causing great damage to the economy, both for breeders who live off the work of these animals and for breeders who invest in the improvement of breeds, preventing access to both national and international markets. The Ministry of Agriculture, Livestock and Food Supply considers AGID to be the official test for diagnosing this disease, but this technique takes time and often ends up being subjective, depending on the particular experience of each laboratory worker. In addition to not being able to detect animals at the beginning of the infection. Therefore, the need to seek new techniques such as indirect ELISA that improve the time of analysis of results, facilitate automation and obtain reliable results. The aim of this study was to standardize an indirect ELISA technique using a GP90 viral envelope protein as an antigen for the diagnosis of equine infectious anemia. Evaluating test performance based on sensitivity, specificity and positive and negative predictive values. The values obtained were 91.11%, 93.33%, 91.11 and 93.33 respectively. It was concluded that the test performs well, in addition to the possibility of detecting positive animals at the beginning of the infection.

Assessment of 3 enzyme linked immunosorbent assays and 1 pseudotype lentivirus-based neutralization test in detecting serum antibody in convalescent plasma from COVID-19 / 中国输血杂志

【Objective】 To assess three severe acute respiratorysyndrome coronavirus 2 (SARS-CoV-2) enzyme linked immunosorbent assays (ELISA) and one pseudotype lentivirus-based neutralization test (ppNAT) in detecting the convalescent plasma antibody levles from COVID-19. 【Methods】 30 COVID-19 convalescent plasma samples were screened for antibodies against SARS-CoV-2 using three kinds of SARS-CoV-2 ELISA reagents and one ppNAT test in Shenzhen. The controls consisted of plasma samples from 32 healthy blood donors in February 2019. The diagnostic efficacy analysis of various SARS-CoV-2 ELISA reagents was performed using real-time fluorescent Polymerase Chain Reaction (RT-PCR). We also analyzed correlation between different immunological reagents and the age, gender, hospitalization, and severity of illness. 【Results】 The positive yielding rate of ppNAT and three kinds of IgG ELISA was higher than that of IgM ELISA. The positive yielding rates of three kinds of IgG ELISA were 100%(30/30), 93.33%(28/30), and 96.67%(29/30) respectively, while the yielding rates in control group were all 0. The positive yielding rate of three IgM ELISAs were 93.33%(28/30), 70%(21/30)and 46.67% (14/30). All the cases from negative control group were negative for IgG and IgM. Pearson correlation coefficient was calculated; there was a strong correlation between ELISA reagent 2 IgG and ELISA reagent 3 IgG (r=0.765, P0.05). 【Conclusion】 In the convalescent plasma with nucleic acid confirmed covid-19, the yielding rates of different IgM antibodies varied greatly. Antibody levels were influenced by age to some extent.

Effect of overexpressed zinc finger protein 580 on apoptosis of oxygen-glucose deprivation SH-SY5Y cells and its possible mechanism / 中华围产医学杂志

Objective:To investigate the expression of zinc finger protein 580 (ZNF580) in oxygen-glucose deprivation (OGD) model of SH-SY5Y cell line and its overexpression on the apoptosis of hypoxic-ischemic neurons and the possible mechanism.Methods:The study was divided into two parts: (1) Human neuroblastoma SH-SY5Y cell line was cultured and divided into the model group and control group. The model group was incubated at 37 ℃ for 6 h in a three-gas incubator of 95% N 2, 5% CO 2, and 0.1% O 2 to establish OGD model, and proteins were extracted at 6, 12, and 24 h after OGD. The expression of ZNF580 was quantified by Western blot. (2) Effects of ZNF580 overexpressed with lentivirus transfection on the apoptosis and cleaved caspase-3 expression: Cells were collected from the control group and model group 24 h after OGD. Overexpressed ZNF580 cells were constructed by lentivirus transfection as the overexpression group and then treated with OGD. Flow cytometry was used to detect the apoptosis rate in the three groups and Western blot was used to detect the expression of cleaved caspase-3. Two independent sample t-test, one-way variance analysis, and LSD- t for pairwise comparison were used for statistical analysis. Results:(1) ZNF580 expression was significantly increased at 6, 12, and 24 h after OGD compared with the control group (1.36±0.05, 2.12±0.07, 1.69±0.05 vs 1.00, LSD- t=9.20, 28.26, and 19.21, all P<0.001). (2) Apoptosis rates of the control, model, and overexpression groups were (1.07±0.56)%, (21.51±1.65)%, and (3.42±0.93)%, respectively, and relative expression levels of cleaved caspase-3 were 1.00, 2.47±0.59, and 1.70±0.25, respectively. Compared with the control group, apoptosis rate and cleaved caspase-3 relative expression level were significantly increased in the model group (LSD- t=21.98 and 8.17, both P=0.001), while the two figures were significantly decreased in the overexpression group when compared with the model group (LSD- t=19.45, P=0.001; LSD- t=4.28, P=0.005). Conclusion:Hypoxia and ischemia could lead to the overexpression of ZNF580, which may reduce the apoptosis of hypoxic-ischemic neurons by inhibiting the expression of cleaved caspase-3 and affecting its enzymatic activation.

Establishment and identification of human immortalized keratinocytes stably expressing human papillomavirus type 16 E6/E7 gene / 中华皮肤科杂志

Objective:To construct human immortalized keratinocytes stably expressing human papillomavirus type 16 (HPV16) E6/E7 gene, and provide a cell model for studying mechanisms underlying HPV16 E6/E7-induced cell immortalization and malignant transformation.Methods:Primary human foreskin keratinocytes (HFKs) were isolated by sequential two-step enzymatic digestion. Cultured HFKs were stably transfected with a HPV16 E6/E7 gene-overexpressing lentiviral vector LV5-HPV16 E6/E7, and consecutively cultured for more than 30 passages. Then, immortalized keratinocytes were screened out and divided into 3 groups: (1) blank control group: second-passage primary HFKs; (2) experimental group: HFKs transfected with LV5-HPV16 E6/E7 at different passages, and the second-passage primary HFKs transfected with LV5-HPV16 E6/E7 were referred to as A0 cells, thereafter, the transfected HFKs were named according to their passage number, such as A1, A2, ... A30; (3) positive control group: the HPV16-positive cervical cancer cell line SiHa. Real-time fluorescence-based quantitative PCR (qRT-PCR) and Western blot analysis were performed to determine the mRNA expression of HPV16 E6/E7 and protein expression of HPV16 E6/E7 and CK14, respectively, in the blank control group, experimental group and positive control group. Cell counting kit-8 (CCK8) assay and Transwell insert invasion assay were conducted to assess the cellular proliferative and invasive activity. In vivo tumor formation experiment in nude mice was conducted to investigate the tumorigenicity of A30 cells in the experimental group and SiHa cells in the positive control group. Results:Primary HFKs were successfully isolated. After the primary HFKs were transfected with the recombinant plasmid LV5-HPV16 E6/E7, the blank control group showed no fluorescence in the cells, but showed senescent phenotypes after serial passages, while in the experimental group, the volume and morphology of A30 cells were similar to those of the primary HFKs with the proportion of fluorescence-positive cells being 100%. Compared with the blank control group, the experimental group showed significantly increased mRNA expression levels of HPV16 E6 and E7 in A1, A10, A20 and A30 cells (HPV16 E6: t = 7.12, 8.07, 6.53, 5.66, P < 0.001, < 0.001, = 0.001, = 0.005, respectively; HPV16 E7: t = 3.20, 4.29, 3.75, 4.22; P = 0.024, 0.008, 0.013, 0.014, respectively) . The protein expression of HPV16 E6/E7 was absent in the blank control group, but was observed in A30 and SiHa cells. CCK8 assay showed that the proliferative activity of A10, A20 and A30 cells was significantly higher than that of the blank control group ( t = 6.49, 7.55, 9.43, P = 0.003, 0.002, 0.001, respectively) , while there was no significant difference in the proliferative activity between A1 cells and the blank control group ( t = 2.40, P = 0.074) . Transwell insert invasion assay showed that A30 cells could not cross the basement membrane, but SiHa cells could pass through the basement membrane and were stained blue. Two months after the inoculation with A30 cells in the nude mice, no visible tumors were found, which was confirmed by a histological study. Subcutaneous tumors were formed in the nude mice after the inoculation with SiHa cells. Conclusion:Human immortalized keratinocytes were successfully established by lentivirus-mediated transfection with HPV16 E6/E7 gene, and can serve as an ideal cell model for HPV-related research.

Effect of miR-204 targeted regulation of DVL3 gene in silica-induced mouse lung epithelial cells / 中华劳动卫生职业病杂志

Objective: To construct a recombinant lentiviral vector for mouse miR-204 overexpression, and to verify the targeted regulation of miR-204 and DVL3 in silica (SiO(2)) -induced mouse lung epithelial cells (MLE-12 cells) . Methods: In October 2019, the pre-miR-204 gene was amplified from the mouse genome by the polymerase chain reaction (PCR) method. After sequencing, the amplified product was cloned into the pLenti-CMV-EGFP lentiviral vector. The positive clones were identified by PCR screening and sequencing. The miR-204 overexpressed lentiviral vector was transfected into 293T cells, and lentiviral packaging and titer determination were performed. The experiment was divided into SiO(2) control group, virus control group, and miR-204 virus group, and the expressions of miR-204 and DVL3 gene were detected by real-time PCR. Results: The miR-204 lentiviral expression vector Lv-miR-204-5p was constructed and identified correctly by PCR and sequencing, and a virus dilution with a titer of 9.57×10(8) IU/ml was obtained. The results of real-time PCR showed that the expression of miR-204 in MLE-12 cells of the miR-204 virus group was higher than that of SiO(2) control group and virus control group, and the expression of DVL3 gene was lower than that of SiO(2) control group and virus control group, the differences were statistically significant (P<0.05) . Conclusion: Overexpression of miR-204 by lentiviral vector may inhibit the expression of DVL3 gene in silica-induced mouse lung epithelial cells.

Effects of N-cadherin silencing on the proliferation and migration of human dental pulp stem cells / 口腔疾病防治

Objective @#To investigate the effects of N-cadherin silencing on the proliferation and migration of human dental pulp stem cells (DPSCs) and to provide experimental evidence for DPSCs-based dental pulp regeneration.@* Methods@# DPSCs were transfected with N-cadherin shRNA lentivirus, and the knockdown efficiency of N-cadherin at both the mRNA and protein levels was confirmed by qRT-PCR and Western blot. The experiment included a negative control group (shRNA -NC) and an N-cadherin shRNA silencing group. Cell proliferation was detected by the CCK-8 method. Cell cycle and apoptosis were assessed by flow cytometry, and cell migration was detected using the Transwell method.@*Results@#N-cadherin shRNA significantly reduced the expression levels of N-cadherin mRNA and protein in DPSCs (P<0.001). The proliferation activity of the N-cadherin shRNA group was significantly greater than that of the shRNA-NC group on the 3rd and 4th days after cell inoculation and lower than that of the shRNA-NC group from the 6th to 8th days (P<0.05). On the 3rd day after cell inoculation, the proportion of cells in S phase and G2 phase in the N-cadherin shRNA group was greater than that in the shRNA-NC group (P<0.05). On the 6th day after cell inoculation, the proportion of cells in S phase and G2 phase in the N-cadherin shRNA group was lower than that in the shRNA-NC group (P<0.05), and the proportion of apoptotic cells in the N-cadherin shRNA group was greater than that in the shRNA-NC group (P<0.01). Low densities cells and high densities cells were inoculated into Transwell upper chamber for 20 h, the number of cells passing through the membrane pores of upper chamber in the N-cadherin shRNA group was greater than that in the shRNA-NC group (P<0.001).@*Conclusion@#Silencing N-cadherin expression can promote the early proliferation and migration of DPSCs.

Optimization of β-globin Stable Expression Using the Third Generation Lentiviral Vector for β-thalassemia Therapy / 中国实验血液学杂志

OBJECTIVE@#To provide a research basis for a safe and effective cell therapy for β-thalassemia through optimization of HS4 region of the third generation lentiviral vector for stable expression of β-globin.@*METHODS@#The human β-globin HS4 region in the third generation lentiviral expression vector was optimized to construct the lenti-HBB, and the transcription and translation of β-globin gene were analyzed by RT-PCR and Western blot after the transduction of lenti-HBB in MEL cell line. Furthermore, the erythroid differentiation of CD34+ cells which were transduced lentiviral virus carrying human β-globin from normal human umbilical cord blood cells and peripheral blood cells of patients with β-thalassemia major were confirmed by colony formation assay, cell smear assay and flow cytometry. The safety and effectiveness of the optimized lenti-HBB were verified by NSG mouse in vivo test.@*RESULTS@#The human β-globin was expressed stably in the MEL cells, and CD34+ cells from health umbilical cord blood as well as PBMC from patient with β-thalassemia major transduced with lenti-HBB could be differentiated to mature red blood cells. The β-globin expression and differentiation in CD34+ cells were demonstrated successfully in the NSG mouse for about 35 months after post-transplant.@*CONCLUSION@#Stable β-globin expression through the optimization of HS4 from CD34+ in the third generation lentiviral vector is safe and effective for patients with severe β-thalassemia and other β-globin abnormal diseases.

Application of RUNX2 gene over expression vector modified exosomes from BMSC combined with calcium carbonate scaffold system in bone defect / 中国骨伤

OBJECTIVE@#To investigate the effect of RUNX2 gene overexpression vector modified exosomes derived from bone marrow mesenchymal stem cells (BMSCs) combined with calcium carbonate scaffold system in bone defect.@*METHODS@#Rabbit BMSCs were used as the research object, and BMSCs were identified by flow cytometry. Construct RUNX2 gene overexpression vector, transfect BMSCs with lentivirus, and collect exosomes by ultracentrifugation. The morphology of exosomes was observed by transmission electron microscope, the expression of exosome marker CD63 was detected by Western blot, and the calcium carbonate scaffold was constructed by three chamber parallel automatic temperature control reaction system. According to whether the RUNX2 gene overexpression vector was transfected or not, the complex of BMSCs and calcium carbonate scaffold was divided into three groups, namely BMSCs group, RUNX2 overexpression group and exosome group. The osteogenic differentiation of BMSCs was detected by oil red O staining and RT-PCR. There were 9 clean adult healthy male New Zealand white rabbits, aged (12.97±1.21) months, with a body weight of (19.3±3.6) kg, with 3 rabbits in each group. The animal model of skull defect was constructed by surgical method, and the repair of bone defect was evaluated by imaging, he staining and Masson staining.@*RESULTS@#The results of flow cytometry showed that the expression of CD29 protein, CD44 protein, CD11b protein and CD45 protein on the surface of BMSCs were 99.5%, 100%, 0.1% and 0.1%, respectively. Transmission electron microscopy showed that the exosomes were bilayer vesicles with a diameter of 50 to 150 nm. Western blot showed that the molecular marker CD63 of exosomes was positive. Oil red O staining showed that the osteogenic differentiation of BMSCs in exosome group was significantly higher than that in RUNX2 overexpression group and BMSCs group. The results of RT-PCR showed that the relative expressions of RUNX2, BMP-2 and ALP mRNA in BMSCs in exosome group were significantly higher than those in RUNX2 overexpression group and BMSCs group (P<0.05). The imaging results showed that the repair effect of skull defect in exosome group was better than that in RUNX2 overexpression group. HE staining and Masson staining showed that the repair effect of skull defect in exosome group was better than that in RUNX2 overexpression group (P<0.05). MSCs in exosome group was significantly higher than that in RUNX2 overexpression group and BMSCs group. The results of RT-PCR showed that the relative expressions of RUNX2, BMP-2 and ALP mRNA in BMSCs in exosome group were significantly higher than those in RUNX2 overexpression group and BMSCs group(P<0.05). The imaging results showed that the repair effect of skull defect in exosome group was better than that in RUNX2 overexpression group. HE staining and Masson staining showed that the repair effect of skull defect in exosome group was better than that in RUNX2 overexpression group(P<0.05).@*CONCLUSION@#Compared with RUNX2 gene overexpression vector transfection, extraction of exosomes directly can promote the differentiation of BMSCs into osteoblasts more efficiently, and the combination with calcium carbonate scaffold can better promote the healing of bone defects. So as to provide new ideas and methods for the clinical treatment of bone defects.

Establishment of SLC6A4 gene silencing stable cell line in PC12 cells and the effect of SLC6A4 gene silencing on apoptosis of hypoxia PC12 cells / 药学实践与服务

Objective To construct SLC6A4-shRNA lentiviral vector, establish PC12 cells stable transformation cell line，and detect the effect of SLC6A4 gene silencing on hypoxia induced PC12 cells apoptosis. Methods Three specific targets sequence of SLC6A4 were designed and short hairpin RNA was synthesized, and then were recombined into shRNA expression vector GV248 plasmid, with non-homology shRNA sequence as negative control. The connection products were switched to competent cells. After dentification and sequencing, the vectors were co-transfected with the auxiliary vectors into 293T cells in order to produce recombinant shRNA lentiviral particles. Then, PC12 cells were infected with the recombinant lentiviral and screened by puromycin. The PC12 cells were divided into two groups: lentiviral negative control group (NC-shRNA) and SLC6A4-silenced group (SLC6A4-shRNA). The expression of SLC6A4 mRNA was detected by real-time fluorescence quantitative and the 5-HTT protein level was assayed by Western blot, The effect of SLC6A4 gene silencing on hypoxia induced apoptosis was detected by flow cytometry. Results The SLC6A4-shRNA lentiviral expression vector was constructed and the recombinant lentiviral particles by packaging the 293T cells were obtained, the stably infected PC12 cells were established after filtering. Compared with negative control group, the expression level of SLC6A4 gene and 5-HTT protein in SLC6A4-shRNA group was suppressed notably (P<0.01). It was confirmed that lentiviral vector could effectively silence SLC6A4 gene in PC12 cells and SLC6A4 gene silencing could decrease apoptosis rate of PC12 cells under hypoxia condition. Conclusion The SLC6A4 gene of PC12 cells could be effectively silenced by shRNA lentivirus vector，which could reverse hypoxia induced apoptosis.

Effect of short hairpin RNA lentivirus-mediated silencing of pleomorphic adenoma gene like-2 on proliferation, migration and invasion of hepatocellular carcinoma cells / 解剖学报

Objective To investigate the effect of short hairpin RNA(shRNA)-mediated pleomorphic adenoma gene like-2 (PALAG2) silencing on the malignant behavior of hepatocellular carcinoma cells and its mechanism. Methods Real-time PCR and Western blotting were used to detect the expression level of PLAGL2 in liver cancer tissues and adjacent tissues. Hepatoma cells MHCC97-L were cultured in vitro, the lentiviral vector plasmid PLAGL2-shRNA and control NC-shRNA were constructed, transfected into MHCC97-L cells, and stable transfected strains were selected with puromycin. CCK-8 and Transwell chamber assay detected the proliferation activity and the number of migration and invasion of MHCC97-L cells after silencing PLAGL2. Western blotting was used to detect the expression of p-PI3K and p-Akt proteins. The PI3K/ Akt signaling pathway activator was used to treat MHCC97-L cells to detect cell proliferation, migration and invasion. Results The expression of PLAGL2 was significantly increased in liver cancer tissue (P < 0. 05). Transfection of 9 strains of MHCC97-L cells with PLAGL2-shRNA could significantly reduce the expression level of PLAGL2, and the ability of proliferation, migration, and invasion of MHCC97-L cells was also weakened (P<0. 05), and the expression levels of p-PI3K, and p-Akt were inhibited (P<0. 05), PI3K/ Akt activator could obviously reverse the above phenomenon. Conclusion shRNA lentiviral vector pathway can effectively silence the expression of PLAGL2 gene in hepatocarcinoma cells. Silencing of PLAGL2 can significantly inhibit the malignant behavior of proliferation, migration and invasion of hepatocarcinoma cells, and its mechanism may be related to the inhibition of PI3K / Akt signaling pathway activation.

Optimization of the Light-On system in a lentiviral platform to a light-controlled expression of genes in neurons

BACKGROUND: Molecular brain therapies require the development of molecular switches to control gene expression in a limited and regulated manner in time and space. Light-switchable gene systems allow precise control of gene expression with an enhanced spatio-temporal resolution compared to chemical inducers. In this work, we adapted the existing light-switchable Light-On system into a lentiviral platform, which consists of two modules: (i) one for the expression of the blue light-switchable transactivator GAVPO and (ii) a second module containing an inducible-UAS promoter (UAS) modulated by a light-activated GAVPO. RESULTS: In the HEK293-T cell line transfected with this lentiviral plasmids system, the expression of the reporter mCherry increased between 4 to 5 fold after light induction. A time expression analysis after light induction during 24 h revealed that mRNA levels continuously increased up to 9 h, while protein levels increased throughout the experiment. Finally, transduction of cultured rat hippocampal neurons with this dual Light-On lentiviral system showed that CDNF, a potential therapeutic trophic factor, was induced only in cells exposed to blue light. CONCLUSIONS: In conclusion, the optimized lentiviral platform of the Light-On system provides an efficient way to control gene expression in neurons, suggesting that this platform could potentially be used in biomedical and neuroscience research, and eventually in brain therapies for neurodegenerative diseases.

Application of CRISPR/Cas9 lentiviral vector in construction of rat hepatic stellate cells with COX-2 gene knockout / 临床肝胆病杂志

ObjectiveTo obtain HSC-T6 cells with stable expression of Cas9 protein and HSC-T6-COX-2-/- cells with COX-2 gene defect by transfecting HSC-T6 cells with CRISPR/Cas9 lentiviral vector, and to provide a good method for further functional research and new strategies for the clinical treatment of liver fibrosis. MethodsThe COX-2 gene-specific sgRNAs (COX-2-sgRNA-1, COX-2-sgRNA-2, COX-2-sgRNA-3) were designed, synthesized, and connected to the GV371 vector, and the recombinant plasmid and the packaging plasmid were transfected into 293T cells to form lentivirus particles; the fluorescence method was used to measure virus titer. The most appropriate amount of the virus was calculated based on MOI. Lenti-Cas9-puro was transfected into HSC-T6 cells, and HSC-T6-Cas9 cells were screened out by puromycin; Lenti-COX-2-sgRNA-EGFP was transfected into HSC-T6-Cas9 cells to obtain HSC-T6-COX-2-/- cells. Cruiser enzyme digestion and Western blot were used to verify gene knockout at the gene and protein levels. An analysis of variance was used for comparison of continuous data between multiple groups, and the least significant difference t-test was used for further comparison between two groups. ResultsSequencing verified that the COX-2-sgRNA expression vector was constructed successfully. Recombinant expression plasmids and packaging plasmids were transfected into 293T cells to form lentivirus particles, and the fluorescence method showed a virus titer of ＞1×108. HSC-T6 cells with stable expression of Cas9 protein and HSC-T6-COX-2-/- cells with COX-2 gene defect were successfully constructed. The HSC-T6-Cas9 group had significantly higher relative mRNA expression of LV-Cas9-Puro than the CON group (541.93±105.76 vs 1.00±0.02, t=12.995, P＜0.01). Cruiser enzyme digestion and Western blot showed that the CRISPR/Cas9 lentivirus expression vectors played a role in the target, among which COX-2-sgRNA-2 knockout had the most significant effect, and this group had a significant reduction in the protein expression level of COX-2 compared with the CON group and the NC group (both P＜0.05), suggesting that COX-2-sgRNA was active. ConclusionA CRISPR/Cas9 lentivirus vector is successfully constructed for COX-2 target gene, and HSC-T6-COX-2-/- cells with stable COX-2 gene knockout are obtained.