Predictive Value of Combined HbA1c and Neutrophil-to-Lymphocyte Ratio for Diabetic Peripheral Neuropathy in Type 2 Diabetes.

BACKGROUND Diabetic peripheral neuropathy (DPN) is a prevalent complication affecting over 60% of type 2 diabetes patients. Early diagnosis is challenging, leading to irreversible impacts on quality of life. This study explores the predictive value of combining HbA1c and Neutrophil-to-Lymphocyte Ratio (NLR) for early DPN detection. MATERIAL AND METHODS An observational study was conducted at the First People's Hospital of Linping District, Hangzhou spanning from May 2019 to July 2020. Data on sex, age, biochemical measurements were collected from electronic medical records and analyzed. Employing multivariate logistic regression analysis, we sought to comprehend the factors influencing the development of DPN. To assess the predictive value of individual and combined testing for DPN, a receiver operating characteristic (ROC) curve was plotted. The data analysis was executed using R software (Version: 4.1.0). RESULTS The univariate and multivariate logistic regression analysis identified the level of glycated hemoglobin (HbA1C) (OR=1.94, 95% CI: 1.27-3.14) and neutrophil-to-lymphocyte ratio (NLR) (OR=4.60, 95% CI: 1.15-22.62, P=0.04) as significant risk factors for the development of DPN. Receiver operating characteristic (ROC) curve analysis demonstrated that HbA1c, NLR, and their combined detection exhibited high sensitivity in predicting the development of DPN (71.60%, 90.00%, and 97.2%, respectively), with moderate specificity (63.8%, 45.00%, and 50.00%, respectively). The area under the curve (AUC) for these predictors was 0.703, 0.661, and 0.733, respectively. CONCLUSIONS HbA1c and NLR emerge as noteworthy risk indicators associated with the manifestation of DPN in patients with type 2 diabetes. The combined detection of HbA1c and NLR exhibits a heightened predictive value for the development of DPN.

[Effect of proximal fibula osteotomy on tension of lateral knee soft tissue in patients with knee osteoarthritis].

OBJECTIVE: To evaluate the short-term efficacy of proximal fibula osteotomy in the treatment of knee osteoarthritis, and to analyze the effect of osteotomy on the tension of the lateral knee soft tissue of patients and verify the reliability of the Arch string theory. METHODS: A total of 71 patients with varus knee osteoarthritis from December 2019 to March 2022 were included, 3 patients dropped out, and 68 patients completed all trials, collected 27 males and 41 females, aged from 51 to 79 years old, with an average of (68.0±7.0 ) years old. The follow-up time ranged from 4 to 12 weeks, with an average of (3.76±1.94) weeks. After admission, the patient underwent Proximal fibula osteotomy, and the tension of lateral knee soft tissue, visual analogue scale (VAS) of pain, the western Ontario and McMaster Universities Osteoarthritis Index (WOMAC) and other indicators were recorded before surgery and 1 month after surgery in the weight-bearing state. RESULTS: According to the VAS, the curative effect of a single index was evaluated by referring to the score before and after treatment by Bao Zongzhao. Thirty seven cases were markedly effective, 27 cases were effective, and 4 cases were ineffective. After surgery, 3 patients presented with weakness of dorsalis pedis extension and 1 presented with paresthesia of dorsalis pedis, which disappeared after symptomatic treatment . The VAS and WOMAC score at 1 month after operation were lower than those before operation, and the differences were statistically significant(P<0.001). The tension of lateral knee soft tissue 1 month after operation was lower than that before operation, and the difference had statistical significance(P<0.001). CONCLUSION: Proximal fibula osteotomy is safe and effective in the treatment of varus knee osteoarthritis in the short term. One month after osteotomy, the tension of lateral knee soft tissue increases under weight-bearing state, but the long-term changes still need further observation and follow-up.

Elevation and phylogeny shape herbaceous seed dormancy in a biodiversity hotspot of southwest China.

Seed dormancy contributes greatly to successful establishment and community stability and shows large variation over a continuous status scale in mountain ecosystems. Although empirical studies have shown that seed dormancy status (SDS) is shaped by elevation and phylogenetic history in mountain ecosystems, few studies have quantified their combined effects on SDS. Here, we collected mature seeds from 51 populations of 11 Impatiens species (Balsaminaceae) along an elevational gradient in the Gaoligong Mountains of southwest China and estimated SDS using mean dormancy percentage of fresh seeds germinated at three constant temperatures (15, 20, and 25°C). We downloaded 19 bioclimatic variables from WorldClim v.2.1 for each Impatiens population and used internal transcribed spacer (ITS), atpB-rbcL, and trnL-F molecular sequences from the GenBank nucleotide database to construct a phylogenetic tree of the 11 species of Impatiens. Logistic regression model analysis was performed to quantify the effects of phylogeny and environment on SDS. Results identified a significant phylogenetic SDS signal in the Impatiens species. Furthermore, elevation and phylogeny accounted for 63.629% of the total variation in SDS among the Impatiens populations. The best logistic model indicated that temperature was the main factor influencing variation in SDS among the Impatiens species, and model residuals were significantly correlated with phylogeny, but not with elevation. Our results indicated that seed dormancy is phylogenetically conserved, and climate drives elevational patterns of SDS variation in mountain ecosystems. This study provides new insights into the response of seed plant diversity to climate change.

Hypoxia-induced FOXO4/LDHA axis modulates gastric cancer cell glycolysis and progression.

BACKGROUND AND AIM: We previously identified forkhead box (FOX) O4 mRNA as a predictor in gastric cancer (GC). However, the underlying mechanism has yet to be elucidated. We aimed to illustrate the mechanism by which FOXO4 regulated glycolysis under hypoxia in GC. METHODS: FOXO4 protein expression was investigated by immunohistochemical staining of 252 GC and their normal adjacent tissues. We restored or silenced FOXO4 expression in GC cell lines to explore the underlying mechanisms. RESULTS: FOXO4 was downregulated in GC. Loss of FOXO4 expression was validated in univariate and multivariate survival analysis as an independent prognostic predictor for overall survival (P < 0.05) and disease-free survival (P<0.05). Restored FOXO4 expression significantly impaired the glycolysis rate in GC cells, while silencing FOXO4 expression enhanced glycolysis rate. FOXO4 expression was inversely associated with maximum standardized uptake value in mice models and patient samples. Mechanistically, FOXO4 bound to the glycolytic enzyme lactate dehydrogenase (LDH)A promoter and inactivated its activity in a dose-dependent manner (P < 0.05). Finally, we determined that FOXO4 was a transcriptional target of hypoxia-inducible factor (HIF) -1α, which is central in response to hypoxia. CONCLUSIONS: Our data suggested that FOXO4 plays a key role in the regulation of glycolysis in GC, and disrupting the HIF-1α-FOXO4-LDHA axis might be a promising therapeutic strategy for GC.

miR-99 inhibits cervical carcinoma cell proliferation by targeting TRIB2.

MicroRNAs (miRNAs) have significant roles in cell processes, including proliferation, apoptosis and stress responses. To investigate the involvement of miR-99 in the inhibition of HeLa cell proliferation, an miR-99 gene expression vector (pU6.1/miR-99), which overexpressed miR-99 in HeLa cells after transient transfection, was constructed. The expression of miR-99 was detected by qPCR. Cell proliferation and apoptosis were analyzed by cell viability, proliferation and apoptosis assays, as well as by electron microscopy. The results showed that overexpression of miR-99 in HeLa cells increased the HeLa cell mortality rate. Moreover, miR-99 overexpression was able to markedly inhibit HeLa cell proliferation according to the 3-(4, 5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide assay. The cell apoptosis rate was significantly higher in pU6.1/miR-99-treated cells compared with that in the control cultures. Increases in intracellular electron density, as well as the proportion of nuclear plasma, blebbing phenomena and apoptotic bodies were observed in pU6.1/miR-99-treated cells compared with control cultures according to electron microscopy analysis. The Tribbles 2 (TRIB2) 3'-untranslated region was also observed to be targeted by miR-99 and the results further demonstrated that miR-99 was able to negatively regulate TRIB2 expression in HeLa cells The results indicate that miR-99 acts as a tumor suppressor gene in HeLa cells, establishing a theoretical basis for its application in cancer therapeutics.

The associations of single nucleotide polymorphisms in miR-146a, miR-196a and miR-499 with breast cancer susceptibility.

BACKGROUND: Previous studies have investigated the association between single nucleotide polymorphisms (SNPs) located in microRNAs (miRNAs) and breast cancer susceptibility; however, because of their limited statistical power, many discrepancies are revealed in these studies. The meta-analysis presented here aimed to identify and characterize the roles of miRNA SNPs in breast cancer risk, and evaluate the associations of polymorphisms in miR-146a rs2910164, miR-196a rs11614913 and miR-499 rs3746444 with breast cancer susceptibility, respectively. METHODOLOGY/PRINCIPAL FINDINGS: The PubMed and Embases databases were searched updated to 31(st) December, 2012. The complete data of polymorphisms in miR-146a rs2910164, miR-196a rs11614913 and miR-499 rs3746444 from case-control studies for breast cancer were analyzed by odds ratios (ORs) with 95% confidence intervals (CIs) to reveal the associations of SNPs in miRNAs with breast cancer susceptibility. Totally, six studies for rs2910164 in miR-146a, involving 4225 cases and 4469 controls; eight studies for rs11614913 in miR-196a, involving 4110 cases and 5100 controls; and three studies of rs3746444 in miR-499, involving 2588 cases and 3260 controls, were investigated in the meta-analysis. The rs11614913 (TT+CT) genotype of miR-196a2 was revealed to be associated with a decreased breast cancer susceptibility compared with the CC genotypes (OR = 0.906, 95% CI: 0.825-0.995, P = 0.039); however, no significant associations were observed between rs2910164 in miR-146a (or rs3746444 in miR-499) and breast cancer susceptibility. CONCLUSIONS: This meta-analysis demonstrates the compelling evidence that the rs11614913 CC genotype in miR-196a2 increases breast cancer risk, which provides useful information for the early diagnosis and prevention of breast cancer.

[Study on the activity against influenza A virus with traditional Chinese medicine Kanggan granules].

OBJECTIVE: To study the anti-influenza A virus effects of traditional Chinese medicine Kanggan granules in chicken embryo and BALB/c mice. METHODS: The influenza A virus (H(1)N(1), FM1) was used in the experiments. FM1 was cultured in chicken embryo and the anti-FM1 activity of Kanggan granules was evaluated through the post-medication hemagglutination titer of FM1. In animal test, 120 healthy BALB/c mice were randomly divided into six groups, normal control, virus control, ribavirin, high-dosage, middle-dosage and low-dosage. The FM1 infection model was established by dripping FM1 into nasal cavity and then the appropriate treatments were prescribed. The effective anti- FM1 indices of Kanggan granules included survival status, protective percentage of death and life elongation percentage of mice infected with FM1. RESULTS: The high and middle doses of Kanggan granules could inhibit the replication of FM1 remarkably in chicken embryo, and could reduced hemagglutination titer to 5 and 3 times. In animal experiments, all mice treated with Kanggan granules could improve the general status of infected mice, the protective percentages of death were 35.0% to 55.0%, the life elongation percentages were 73.0% to 88.9% and the minimal effective dose was 3.00 g/kg. CONCLUSION: Kanggan granules can inhibit the replication of influenza A virus and protect the mice infected with FM1.

[Study on the anti-NTHi infection of Hap recombinant protein in vivo].

AIM: To observe the immune effect of Hap recombinant protein on murine model of bronchopneumonia infected with NTHi, and explore the mechanism about the anti-NTHi infection. METHODS: The C57BL/6 mice intranasally immunized with purified Hap recombinant protein and CT-B were challenged by NTHi encased in agar beads. The immunifaction of anti-infection was observed through encocyte counting of BALF, bacteria detection of lung and the pathologyical change of lung tissue. RESULTS: In the challenge with NTHi experiment, the inflammatory exudation of the infected murine and pathological change of lung tissue was relieved by combined immunization of Hap recombinant protein and CT-B, and quantity of NTHi in lung of the infected murine was reduced obviously. CONCLUSION: The Hap recombinant protein also had good ability of anti-NTHi infection in the murine model of NTHi bronchopneumonia. This study could offer the oretical and experimental basis for development of new vaccine against NTHi.

[Effect of danshensu on activation of JNK pathway in hepatic stellate cells(HSCs) induced by IL-1 beta].

OBJECTIVES: To investigate the inhibitory effect of danshensu on the activation of JNK signaling in rat hepatic stellate cells (HSCs) induced by IL-1beta. METHODS: The proliferation of primary rat HSCs treated with different concentration of Danshensu was checked by MTT colorimetric assay. The expression and phosphorylation of JNK and P-JNK was detected by western blotting. Synthesis and secretion of collagen I were detected by the quantitative immunocytochemical assay and ELISA. RESULTS: Danshensu inhibited the proliferation of HSCs in a dose-dependent manner. At the concentration of 0.0625 to 0.25 mmol/L, Danshensu significantly repressed the proliferation of HSC induced by IL-1beta (P < 0.05). Synthesis and secretion of Type I collagen was significantly decreased 24 hours after 0.25 mmol/L Danshensu treatment (P < 0.01). The phosphorylation of JNK induced by IL-1beta was significantly inhibited by Danshensu treatment (P < 0.05), however, the expression of JNK was not regulated by Danshensu. CONCLUSION: Danshensu represses the activation and proliferation of HSCs, and inhibits the synthesis and secretion of Type I collagen, possibly via the repression of the JNK signal transduction.

[Construction of a eukaryotic expression vector of the gene encoding rat interferon-gamma-inducible protein and its expression in NIH 3T3 cells].

OBJECTIVE: To construct an expression vector of the gene encoding rat interferon-gamma-inducible protein (IP-10) and identify its expression in NIH 3T3 cells. METHODS: IP-10 gene was amplified by polymerase chain reaction (PCR) and inserted into the eukaryotic expression vector pcDNA3.1(+). After identification by PCR, restriction endonuclease digestion and sequence analysis, the recombinant expression vector pcDNA3.1(+)-IP-10 was transfected into NIH 3T3 cells via liposome. Immunofluorescence method was used to confirm the expression of pcDNA3.1(+)-IP-10 in the transfected NIH 3T3 cells. The expression of IP-10 protein in the supernatant of the transfected cells was examined by Western blotting. RESULTS: PCR, restriction endonuclease digestion and sequence analyses confirmed successful construction of the recombinant vector pcDNA3.1(+)-IP-10. Immunofluorescence assay identified the expression of pcDNA3.1(+)-IP-10 in NIH 3T3 cells, and the expression of IP-10 protein was detected by Western blotting in the supernatant of the transfected cells. CONCLUSION: A eukaryotic expression vector pcDNA3.1(+)-IP-10 has been successfully constructed, which provides the basis for investigating the therapeutic effect of IP-10 on Th1 type autoimmune disease.

[Construction and expression characteristics in vitro of human papillomavirus type 16 E7 suicidal DNA vaccine].

OBJECTIVE: To construct the suicidal DNA vaccine of human papillomavirus type 16 E7 gene (HPV16), and explore the DNA vaccine expression characteristics in vitro and capacity of inducing the transfected cells into apoptosis. METHODS: HPV16 E7 gene cloned by PCR from pET32/E7 was inserted into the plasmid pSCA1 to construct the recombinant plasmid pSCA/E7, followed by identification with PCR, BamH I and Sma I digestion and sequencing. pSCA/E7 was then used to transfect BHK-21 cell line. The transient expression of HPV16 E7 gene was confirmed by immuno-fluorescent staining, and the apoptosis induced by pSCA/E7 was checked with TDT-mediated dUTP nick end-labeling (TUNEL). RESULTS: The cloned E7 gene fragment was about 400 bp in length. PCR, restriction endonuclease digestion and sequence analysis revealed that the HPV16 E7 gene was cloned into the eukaryotic expression plasmid pSCA1 successfully. Immunofluorescent staining confirmed that the E7 gene could express in BHK-21 cell line. The BHK-21 cells transfected with pSCA/E7 could be induced into apoptosis which was confirmed by TUNEL. CONCLUSION: The results show that HPV16 E7 suicidal DNA vaccine can express in BHK-21 cell line, and induce the pSCA/E7 transfected cells into apoptosis. These findings may provide the foundation for exploring the therapeutic vaccine against HPV16-associated cervical cancer.

[Construction of adenovirus expressing siRNA against hTR and its anti-tumor activity in vitro].

AIM: To study the effect of the hTR-siRNA adenovirus on hTR mRNA gene silence, telomerase activity inhibition and anti-tumor in vitro. METHODS: RNAi adenovirus vector, Ad-hTR-siRNA, and negative control Ad-NT-siRNA were constructed by an improved ligation method. Different tumor cells and liver cell line, HL-7702, were infected with 100 MOI of the recombinant adenoviruses. TRAP-ELISA, Real-time PCR and FCM were used to analyze telomerase activity, hTR mRNA, apoptosis rate and hTERT protein expression. RESULTS: As compared with Ad-NT-siRNA, Ad-hTR-siRNA reduced both hTR mRNA levels (70.21%) and telomerase activity (58.87%) of HeLa cells significantly, increased apoptosis rate (29.7%). But the telomerase activity of HL-7702 and hTERT protein didn't show the tendency of decrease. CONCLUSION: It is supposed that the hTR-siRNA adenovirus could knockdown hTR gene specifically and suppress the tumor cell growth in vitro efficiently. Maybe this siRNA expressing recombinant adenovirus system could be a new method for cancer gene therapy.

[Expression and immunity of fused protein H1N1 M2e and cholera toxin B].

AIM: To construct the eukaryotic recombinant plasmid pcDNA3.1a(+)-M2e/CtB which contains H1N1 M2e gene and cholera toxin B subunit gene (CtB) and to study the expression and immunity of recombinant protein M2e/CTB in NIH3T3 cells. METHODS: M2e/CtB gene was cloned by PCR and digested with Hind III and Xho I. M2e/CtB was linked into pcDNA3.1a(+) to build eukaryotic expression plasmid pcDNA3.1a(+)-M2e/CtB. The pcDNA3.1a(+)-M2e/CtB was transfered into competent E.coli JM109. The transformed colonies were verified by Hind III and Xho I, PCR, and sequencing. The NIH3T3 cells were transfected with pcDNA3.1a(+)-M2e/CtB by positive ion polymer. The product of M2e/CtB gene with stable expression was analysed by immunofluorescence, RT-PCR sequencing, and Western blot. RESULTS: The digested pcDNA3.1a (+)-M2e/CtB contains correct M2e and CtB gene. Blastn results showed the genetic homongenicity of M2e and CtB were 100%, respectively. The pcDNA3.1a(+)-M2e/CtB efficiently expressed M2e/CTB protein in the eukaryotic NIH3T3 cells. The cell lysis and supernant both contained the M2e/CTB protein, which had the immunity and reactivity to both anti-M2e and anti-CTB. The relative molecular mass of M2e/CTB protein was about 18 000 analysed by Western blot. CONCLUSION: A new recombinant eukaryotic plasmid pcDNA3.1a(+)- M2e/CtB has been successfully constructed. The plasmid can express the fused protein of M2e with CTB. Our study will help further research into new and effective DNA vaccines against influenza virus A.

[Optimized isolation and purification of non-typeable Haemophilus influenzae Haps protein].

OBJECTIVE: To optimize the isolation and purification conditions for Hap(s) protein of non-typeable Haemophilus influenzae. METHODS: Hap(s) protein was purified by ammonium sulfate precipitation, dialysis desalting and Hitrap weak cation exchange columns of CM Sepharose Fast Flow. The condition of the elution was optimized for pH and ionic strength, the absorbance at 280 nm of the elution samples were detected, and the targeted protein band in the collected samples was observed by SDS-PAGE electrophoresis. RESULTS: The Hitrap ion exchange column was eluted with buffer 1, which resulted in a baseline distribution of absorbance at 280 nm. Buffer 2 elution of the column resulted in the presence of peak absorbance with trails, which was identified to be constituted by some low molecular weight bands by subsequent SDS-PAGE. In serial column elution with buffer 3 with different ionic strength, a peak absorbance was observed with the ionic strength of 100 mmol/L NaCl, and SDS-PAGE confirmed that the peak was generated by the target protein. No obvious peaks or bands in SDS-PAGE occurred with the other ionic strengths. CONCLUSION: The pH of the buffer only affect the elution of the irrelevant proteins rather than the Hap(s) protein, and elution with the buffer containing 100 mmol/L NaCl can be optimal for eluting the Hap(s) protein.

[Therapeutic effect of a new recombinant immunotoxin mMIP-1alpha-DT390 on experimental autoimmune encephalomyelitis].

OBJECTIVE: To evaluate the therapeutic effect of a new recombinant immunotoxin mMIP-1alpha-DT390 on experimental autoimmune encephalomyelitis (EAE). METHODS: EAE was induced in the low-sensitive strain C57BL/6 mice with intraperitoneal injection of myelin basic protein (MBP) to simulate the human disease multiple sclerosis, followed by intramuscular injection of cationic liposome carrying the plasmid DNA SRalpha-mMIP-1alpha-DT390 in the leg muscle to elicit resistance to EAE development. The mice were then examined daily for clinical signs of EAE by an observer blind to the treatment protocol. For immunohistochemistry the mice were anesthetized and perfused with sterile PBS and paraformaldehyde, and the cerebrum, cerebellum, medulla and spinal cord were removed for preparation of serial sections. The mononuclear cells (MNCs) from the EAE mouse spleens were prepared for three-color flow cytometry analysis of the surface markers with appropriate antibodies following the BD Pharmingen cytokine staining protocol. RESULTS: EAE model was successfully established by active MBP immunization in C57BL/6 mice. Administration of the immunotoxin mMIP-1alpha-DT390 significantly delayed the disease onset and lowered the mean clinical score for EAE as compared with the control mice. Immunohistochemistry demonstrated much less CCR5(+) infiltrating cells in the central nervous system in mMIP-1alpha-DT390-treated mice than in the control. The treatment also eliminated reactive T cells in the periphery blood without affecting the number of B cells. CONCLUSION: The immunotoxin mMIP-1alpha-DT390 can attenuate the disease activity of EAE in mice, suggesting its potential use in the treatment of other autoimmune disorders.

[Effects of ST6Gal I antisense oligonucleotide-mediated gene silencing on cell adhesion and invasiveness of hela cells].

OBJECTIVE: To study the effects of antisense oligonucleotide (ASODN) targeting ST6Gal I on cell adhesion and invasiveness of human cervical carcinoma cell line HeLa which over-expressed ST6Gal I . METHODS: ASODN and sense oligonucleotide (SODN) targeting ST6Gal I were designed and constructed, and transfected into a cervical cancer cell line, HeLa, by lipofectmine 2000. HeLa cells were cultured and divided into 4 groups: blank control group, liposome group, SODN group and ASODN group. RT-PCR was used to examine the ST6Gal I mRNA expression. Flow cytometry was used to examine the amount of alpha2, 6-sialylation on the HeLa cell surface. The HeLa cell adhesion and invasiveness to extracellular matrix ( ECM) were analyzed by using CytoMatrixTM kit and cell invasion assay kit, respectively. RESULTS: The expression of ST6Gal I mRNA in HeLa cells at 48 hrs after transfection in the ASODN group was significantly decreased in comparison with that in the blank control group, liposome group, and SODN group(P <0. 01). The amount of alpha2,6-sialylation on cell surface in ASODN group was significantly lower than that of the other 3 groups ( P <0. 05). The adhesion and invasiveness of the cells in the ASODN group decreased remarkably, both significantly lower than those of the other 3 groups ( all P < 0. 05). CONCLUSION: Specific ASODN targeting ST6Gal I effectively inhibits HeLa cell ST6Gal I expression, decreases the amount of alpha2,6-sialylation on cell surface and leads to a decline of cell adhesion and invasiveness to ECM. This result also established a fine base for further studying on anti-tumor treatment with antisense oligonucleotide.

[Combinatorial effects of ST6Gal I siRNA and antisense oligonucleotide-mediated gene silence on metastasis ability of cervical carcinoma cells].

OBJECTIVE: To study the combinatorial effects of small interfering RNA (siRNA) and antisense oligonucleotides (ASOs) targeting alpha 2,6 sialyltransferase (ST6Gal I) on the cell adhesion and invasiveness of human cervical carcinoma cell line, HeLa with over-expressing ST6Gal I. METHODS: The siRNA and ASOs targeting to ST6Gal I were designed and synthesized chemically, and then with lipofectamine 2000, transferred into HeLa cells, which were cultured and divided into 7 groups: blank control group, liposome group, siRNA group transfected with ST6Gal I siRNA, ASO1 group transfected with ST6Gal I ASO whose target site is same as siRNA, ASO2 group transfected with ST6Gal I ASO whose target site is different with siRNA, siRNA+ASO1 group transfected with siRNA and its homologous ASO1, siRNA+ASO2 group transfected with siRNA and its nonhomologous ASO2. RT-PCR was used to examine the ST6Gal I mRNA expression. Flow cytometry was used to examine the amount of alpha2, 6-sialylation on the HeLa cell surface. The HeLa cell adhesion or invasiveness to extracellular matrix (ECM) was analyzed by using CytoMatrix kit or cell invasion assay kit, respectively. RESULTS: The expression of ST6Gal I mRNA, the amount of a2,6-sialylation of cell surface, the cell adhesion and invasion to ECM were remarkably decreased in siRNA group, ASO1 group, ASO2 group, siRNA+ASO, group and siRNA+ASO2 group, and all significantly lower than those in the blank control group and liposome group (all P < 0.05), especially in siRNA+ASO2 group. The difference was significant between siRNA+ASO2 group and siRNA group (all P < 0.05). No significant difference was observed between siRNA+ASO1 group and siRNA group, neither between blank control group and liposome group (all P > 0.05). CONCLUSION: The synthesized chemically specific siRNA targeting to ST6Gal I can effectively downregulate the ST6Gal I expression in HeLa cell and further lead to the decline of cell adhesion and invasiveness to ECM, and use of associating siRNA with ASO targeting to same gene but different oligonucleotide location possesses the synergy and additive effect on targeted gene expression knocked down.

[Steady expression and activity determination of recombinant signal peptide of mBD2 in SiHa cell].

OBJECTIVE: To observe the expression pattern and effect of recombinant murine beta defensin 2 (rmBD2) on the proliferation of cell transfected with pcDNA3. 1 (+)/rmBD2. METHODS: The recombinant plasmid pcDNA3. 1 (+)/rmBD2 was transferred into SiHa cells. The transfected SiHa cells were selected by G418 with 100 microg/mL for over 20 days. The steady expressions of rmBD2 protein and rmBD2 mRNA were detected by immunofluorescence and RT-PCR. The effects of rmBD2 on SiHa cell growth and reproduction were measured by MTT. RESULTS: The SiHa cells which could stably express the rmBD2 protein were harvested with 100 microg/mL of G418. The rmBD2 protein expression was, by immunofluorescence, confirmed and its mRNA in SiHa with rmBD2 could be detected at 4 weeks, 6 weeks, 8 weeks, and 10 weeks after cell transfected. A 220 bp segment encoding rmBD2 was amplified by RT-PCR and proved by sequencing. The SiHa/K cells transfected pcDNA3. 1 (+) and SiHa had no expression of rmBD2 protein. The SiHa with rmBD2 expression grew slowly than SiHa/K and SiHa control (P < 0.05). CONCLUSION: The screening for SiHa with rmBD2 expressing stably is successful. The cell growth curve shows that rmBD2 can inhibit the proliferation of tumor cells. The above results establish a solid foundation for further studying the biological properties and the anti-tumor mechanism of beta defensins.

[ST6Gal I siRNA and antisense oligonucleotide-mediated gene silencing lowers the invasiveness potential of colonic carcinoma cells].

OBJECTIVE: To study the effects of small interfering RNA (siRNA) and antisense oligonucleotides (ASOs) targeting ST6Gal I on adhesion and invasiveness of human colonic carcinoma cell line SW480 over-expressing ST6Gal I. METHODS: siRNA and ASOs targeting ST6Gal I were constructed and transfected into SW480 cells via lipofectmine 2000. SW480 cells were cultured and divided into 7 groups, namely the blank control group, liposome group, siRNA group (transfected with ST6Gal I siRNA), ASO(1) group (transfected with ST6Gal I ASO whose target site is different from the siRNA), ASO(2) group (transfected with ST6Gal I ASO targeting the same site as siRNA), siRNA+ASO(1) group (transfected with siRNA and ASO(1)), siRNA+ASO(2) group (transfected with siRNA and ASO(2)). RT-PCR was used to examine ST6Gal I mRNA expression following the treatment. Flow cytometry was used to examine the amount of alpha2,6-sialylation on SW480 cell surface. SW480 cell adhesion and invasiveness to the extracellular matrix (ECM) were analyzed using CytoMatrix kit and cell invasion assay kit, respectively. RESULTS: The expression of ST6Gal I mRNA, the amount of alpha2,6-sialylation on the cell surface and cell adhesion and invasion to ECM decreased remarkably in groups siRNA, ASO(1), ASO(2), siRNA+ASO(1) and siRNA+ASO(2), all significantly lower than those of the blank control and liposome groups (all P<0.05), especially in siRNA+ASO(1) group. Significant difference was noted between siRNA+ASO(1) and siRNA groups (P<0.05), but not between siRNA+ASO(2) and siRNA groups, or between blank control and liposome groups (all P>0.05). CONCLUSION: Chemically synthesized specific siRNA targeting ST6Gal I effectively inhibits SW480 cell ST6Gal I expression and leads to diminished cell adhesion and invasiveness to ECM, suggesting a combined effect of siRNA and ASO with different targeting sites.

[Primary study of the recombinant immunotoxin DT390-mRantes in EAE therapy].

AIM: To construct a novel eukaryotic expression plasmid including the recombinant immunotoxin DT390-mRantes and treat experimental autoimmune encephalomyelitis (EAE) in mice. METHODS: EAE in C57BL/6 mice were induced by the extracted MBP. The mRantes fragment was inserted into the eukaryotic expression plasmid SRalpha containing DT390. Then cationic liposome-embedded plasmid DNA was injected into the muscles of the hind-limbs in mice. The effect of DT390-mRantes was evaluated by observing clinical symptoms, pathological changes of brain, relative cytokine of peripheral blood, and the proportion of T cells and B cells. RESULTS: The recombinant immunotoxin DT390-mRantes was successfully constructed. Compared the mice in treated group with those in untreated group the clinical symptoms of EAE were alleviated, the infiltration of inflammatory cells were decreased, the IFN-gamma level was fallen, and the ratio of T/B cells was decreased. CONCLUSION: The recombinant immunotoxin DT390-mRantes has distinct effects on EAE in mice, which may be used for beneficial reference to the therapy of MS.