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Objective:To study the characteristics and clinical significance of mitochondrial injury of T lymphocyte subsets in patients with autoimmune hepatitis (AIH).Methods:The clinical data of 57 patients with AIH (AIH group) from June to December 2021 in Hangzhou Xixi Hospital were retrospectively analyzed, while 60 healthy physical examiners were included as healthy group. The peripheral blood T lymphocyte subsets (CD 8+ T lymphocyte count and CD 4+ T lymphocyte count) were detected by flow cytometry, and matched mitochondrial staining value according to certain algorithm was used to determine the mitochondrial damage of helper T lymphocyte (Th cell) and suppressor T lymphocyte (Ts cell). The levels of IgG, alanine aminotransferase (ALT) and aspartate aminotransferase (AST) were measured using a Roche E170 automatic electrochemiluminescence immunoassay. Anti-nuclear antibody (ANA) titer was measured by immunofluorescence. Multivariate Logistic regression was used to analyze the independent risk factors of mitochondrial damage of Th cell and Ts cell in patients with AIH. Results:The ALT, AST, IgG, positive rate of ANA titer, CD 4+ T lymphocyte count, CD 8+ T lymphocyte count, rate of Th cell mitochondrial injury and rate of Ts cell mitochondrial injury in AIH group were significantly higher than those in healthy group: (118.90 ± 37.61) U/L vs. (30.96 ± 14.37) U/L, (102.40 ± 36.51) U/L vs. (31.12 ± 14.06) U/L, (18.40 ± 3.71) g/L vs. (13.89 ± 1.98) g/L, 96.49% (55/57) vs. 16.67% (10/60), 438 (323, 637) × 10 6/L vs. 398 (272, 469) × 10 6/L, 296 (211, 296) × 10 6/L vs. 270 (193, 322) × 10 6/L, 61.40% (35/57) vs. 8.33% (5/60) and 82.46% (47/57) vs. 11.67% (7/60), and there were statistical differences ( P<0.01 or <0.05). Multivariate Logistic regression analysis result showed that the AST elevated and CD 8+ T lymphocyte count reduced were the independent risk factors of Ts cell mitochondrial injury in patients with AIH ( OR = 1.06 and 0.99, 95% CI 1.01 to 1.10 and 0.99 to 1.00, P<0.05); the ALT elevated and IgG elevated were the independent risk factors of Th cell mitochondrial injury in patients with AIH ( OR = 1.08 and 1.66, 95% CI 1.02 to 1.14 and 1.11 to 2.48, P<0.05). Conclusions:It is of positive clinical significance to measure the T lymphocyte subtype mitochondrial injury in patients with AIH. The probability of mitochondrial injury of T lymphocyte subtype can be predicted by biochemical indexes such as ALT, AST and IgG, so as to indirectly evaluate the liver cell necrosis.
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OBJECTIVE@#In this study, the role and potential mechanism of transformer 2β (Tra2β) in cervical cancer were explored.@*METHODS@#The transcriptional data of Tra2β in patients with cervical cancer from Gene Expression Profiling Interactive Analysis (GEPIA) and cBioPortal databases were investigated. The functions of Tra2β were evaluated by using Western blot, MTT, colony formation, Transwell assays, and nude mouse tumor formation experiments. Target genes regulated by Tra2β were studied by RNA-seq. Subsequently, representative genes were selected for RT-qPCR, confocal immunofluorescence, Western blot, and rescue experiments to verify their regulatory relationship.@*RESULTS@#The dysregulation of Tra2β in cervical cancer samples was observed. Tra2β overexpression in Siha and Hela cells enhanced cell viability and proliferation, whereas Tra2β knockdown showed the opposite effect. Alteration of Tra2β expression did not affect cell migration and invasion. Furthermore, tumor xenograft models verified that Tra2β promoted cervical cancer growth. Mechanically, Tra2β positively regulated the mRNA and protein level of SP1, which was critical for the proliferative capability of Tra2β.@*CONCLUSION@#This study demonstrated the important role of the Tra2β/SP1 axis in the progression of cervical cancer in vitro and in vivo, which provides a comprehensive understanding of the pathogenesis of cervical cancer.
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Humans , Animals , Mice , Female , Uterine Cervical Neoplasms/genetics , HeLa Cells , Cell Proliferation , Biological Assay , Transcription Factors , Sp1 Transcription Factor/geneticsABSTRACT
Due to the characteristics such as high capture, high recovery and precise control with fluid, the microfluidic chip has attracted much attention in the research field of circulating tumor cells (CTCs). The developed microfluidic system mainly included three types based on the captured principles such as biological affinity tag microfluidic chip, free label microfluidic chip and rely on biological affinity with the physical properties of integrated microfluidic chip.
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Objective@#The underlying mechanism of Ezrin in ovarian cancer (OVCA) is far from being understood. Therefore, this study aimed to assess the role of Ezrin in OVCA cells (SKOV3 and CaOV3) and investigate the associated molecular mechanisms.@*Methods@#We performed Western blotting, reverse transcription-quantitative polymerase chain reaction, MTT, cell colony, cell wound healing, transwell migration and invasion, RhoA and Rac active pull down assays, and confocal immunofluorescence experiments to evaluate the functions and molecular mechanisms of Ezrin overexpression or knockdown in the proliferation and metastasis of OVCA cells.@*Results@#The ectopic expression of Ezrin significantly increased cell proliferation, invasiveness, and epithelial-mesenchymal transition (EMT) in OVCA cells. By contrast, the knockdown of endogenous Ezrin prevented OVCA cell proliferation, invasiveness, and EMT. Lastly, we observed that Ezrin can positively regulate the active forms of RhoA rather than Rac-1 in OVCA cells, thereby promoting robust stress fiber formation.@*Conclusion@#Our results indicated that Ezrin regulates OVCA cell proliferation and invasiveness by modulating EMT and induces actin stress fiber formation by regulating Rho-GTPase activity, which provides novel insights into the treatment of the OVCA.
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Female , Humans , Cell Line, Tumor , Cell Movement , Cell Proliferation , Cytoskeletal Proteins/metabolism , Epithelial-Mesenchymal Transition , Gene Expression Regulation, Neoplastic , Neoplasm Invasiveness , Ovarian Neoplasms/pathology , Stress Fibers/metabolism , rhoA GTP-Binding Protein/metabolismABSTRACT
Objective:To compare the consistency and detection capability of seven 2019-nCoV nucleic acid detection kits, and provide reference for detection method selection of clinical laboratory and diagnosis of new coronavirus pneumonia.Methods:Two batches of pharyngeal swab samples were collected from tenpatients with confirmed infection of 2019-nCoV and 10 suspected patients with negative 2019-nCoV test results during January 29 to February 5, 2020 in Shenzhen Luohu People′s Hospital. Seven kinds of kits were labeled as ato g and used for nucleic acid detection respectively to evaluate the consistency of the test results of the clinical samples. A 2019-nCoV positive specimen was selected and diluted to 5-concentration gradient plates (Level-1 to 5) with RNase-free water. The positive detection rate and intra-batch repeatability of different brands of kits were compared.Results:The negative and positive coincidence rates of twenty clinical samples tested by six kinds of kits were 100%, and the positive and negative coincidence rate was 8/10 and 10/10 for the other kit, respectively. The results of intra-batch repeatability showed the CVs of viral loads tested by these seven kits were all less than 5%. In the concentration range of Level-1 to 3, the detection capability for open reading frame (ORF)1ab gene of Kit b,d and f was lower than Kit a,c,e and g, and the detection capability of kit e and g was the highest (14/15). The detection capability for N gene of Kit a (15/15) was higher than the other 5 kits. The comprehensive analysis of the detection capability for ORF1ab and N gene showedthat Kit d had the lowest detection capability (ORF1ab:40%,N:53%), and there was no significant difference in the detection capability of Kit a, b, c, e, and f.Conclusions:There was no significant difference in the accuracy and repeatability of the seven kits for positive samples with high viral loads, and the detection performance was good; but some kits had poor detection capability for weak positive samples. It is suggested that the weak positive samples should be rechecked by at least two manufacturers′ kits to ensure the accuracy of the results.
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Objective Non-muscle myosin heavy chain ⅡA (NMHC ⅡA ) plays a significant role in tumor progression and metastasis .Our prior study showed that the expression of NMHC ⅡA was much higher in human bladder cancer sample than that in adjacent tissue .The increased level of NM HC ⅡA expression was correlated with worse prognosis .However ,the role of NMHC ⅡA is unknown in the invasion and metastasis of bladder cancer .Methods RT-PCR and western blotting were used to examine NMHC ⅡA expression lev-els in normal bladder epithelial cells and bladder cancer cell lines .T he migration and invasion ability of cells was tested by wound healing assay and Transwell invasion assay ,respectively .Results Our study showed that knockdown of NMHC ⅡA inhibited migration and invasion in bladder cancer cell line .Conclusion The study indicated that NM HC ⅡA expression increased the invasion and metastasis ability of bladder cancer cell line in vitro .
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Objective To examine the availability of emergency medical services(EMS) for patients with acute stroke and to investigate influential factors affecting the preference of patients'to EMS.Methods Consecutive information of patients with acute stroke who presented to the emergency department of Zhongnan Hospital of Wuhan University from June 1, 2014 to December 31, 2015 were analyzed.Gender, age of patients, transport modality, risk factors in cerebrovascular diseases, initial symptoms, stroke types, onset and admission time were included to make analysis in retrospective study.The participants were divided into two groups based on the preference of patients, namely EMS group and self-transport group.Wilcoxon rank sum test or Chi-squared test was used to statistical analysis as appropriate.A multiple binary logistic regression was used to determine the relationship between various patient-related factors with probability whether patients chose EMS or not.Results Among the 240 patients, only 29.2%of them asked for the EMS at the onset of symptoms (EMS group, n=70), and the rest patients called for other services(self-transport group, n=170).Logistic regression analysis revealed that the patients with the symptom of altered consciousness or convulsion (95%CI:0.107-0.403,OR=0.208,P<0.01) were more likely to use EMS.The time consumed from onset to visit of patients with acute stroke to emergency physician was significantly shorter in EMS group (M, 60 min vs.180 min,P<0.01).Conclusion The symptom of altered consciousness or convulsion was the independent factor to determine whether patients with acute stroke preferred EMS or not.
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Objective To establish a method to research the fingerprint and determine the ingredients of Saxifraga stolonifera by HPLC, which aimed at providing reference for the quality control of Saxifraga stolonifera. Methods Agilent Eclipse XDB-C18 (150 mm × 4.6 mm, 5 μm) column was used as the stationary phase, and the mobile phase consisted of acetonitrile (A)-0.1% phosphoric acid with gradient elution at the flow rate of 1.0 mL/min. The detection wavelength was 254 nm, and the column temperature was maintained at 35 ℃. The result would be analyzed by SOP of Similarity evaluation system for chromatographic fingerprint of TCM. Results The results obtained by the method of fingerprint analysis are good. In the fingerprints, 11 peaks were selected as the common peaks to evaluate the similarities among 21 batches of S. stolonifera collected from different regions, and five contents of them were indentified as protocatechuic acid, gallic acid, bergenin, quercetin-5-O-β-D-glucoside, and quercitrin. The similarities between standard herb and each determined herb showed a lot of differences from others. The determination method showed that there weregood linear relationships of five figured contents in the range of 0.052 8-0.844 8, 0.020 96-0.335 36, 0.241 6-3.865 6, 0.130 8-2.092 8, and 0.023 68-0.378 88 μg. Moreover, the recoveries rates of five figured contents are 96.64%, 100.72%, 96.62%, 103.71%, 96.75%,and all RSDs were lower than 2%. The contents of 5 components in the gathered herbs were determined by external standard method in the range of 0.07-0.40, 0.19-4.36, 1.42-5.98, 0.42-6.86, and 0.11-1.51 mg/g. Conclusion The method established in this study is simple, reliable and durable, which can provide a scientific basis for the quality control of S. stolonifera.
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Objective To investigate the clinical significance of serum total bile acid(TBA)and cholyglycine(CG)detection in the early diagnosis of intrahepatic cholestasis of pregnancy(ICP)and perinatal adverse outcomes.Methods Chose 67 ca-ses of ICP pregnant women diagnosed and treated in Chang'an Hospital from June 2015 to June 2017 and they were selected as observation group.According to the 2015 edition of the diagnostic guidelines for the diagnosis and treatment of intrahe-patic cholestasis of pregnancy.The patients were divided into mild ICP group and severe ICP group,and 60 healthy pregnant women were selected as the control group.The serum TBA concentration was measured by fifth generation cyclic enzyme method and the concentration of serum CG was detected by latex enhanced turbidimetric immunoassay.The serum TBA,CG test results and the rate of abnormal test results,the incidence rate of perinatal adverse outcomes were compared between groups.Evaluation of serum TBA and CG detection of pregnancy early diagnosis of intrahepatic cholestasis and clinical value of perinatal adverse outcomes.Results The detection results of serum TBA and CG in the control group,mild ICP group and severe ICP group,there were significant differences between the three groups,the difference was statistically significant (P<0.01),the detection results in the CG group,serum TBA,ICP slightly higher than the control group,the difference was statistically significant(t=22.27,39.68,P<0.05).Weight of serum TBA and ICP group,the results of CG was higher than that of patients with mild ICP group,the difference was statistically significant(t=10.24,70.87,P<0.05).And in the con-trol group,mild ICP group,severe ICP group pregnant women serum TBA,CG test results increased with the aggravation of the disease.Serum TBA and CG abnormal results in 60 cases of the control group were not detected.In 67 cases of group ICP(mild ICP group and severe ICP group)were 63 cases and 61 cases,two groups of abnormal results rate comparison,and the difference was statistically significant(χ2=29.35,31.27,P<0.01).Perinatal premature labor,fetal distress,perinatal death and stillbirth incidence of adverse perinatal outcomes in the control group,mild ICP group and severe ICP group were significantly different between the three groups(χ2=39.17,56.31,13.02,6.92,P<0.01).Conclusion Intrahepatic chole-stasis of pregnancy,serum TBA and CG increased significantly,can be used as a sensitive indicator of ICP diagnosis,improve the detection rate of ICP,and effectively predict perinatal outcome.For intrahepatic cholestasis of pregnancy early detection and early diagnosis,it has important clinical significance.
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A tcpS-based PCR method was established to simultaneously screen Salmonella enterica serovars Enteritidis,Pullorum/Gallinarum,and Dublin.The developed PCR method provides laboratorial support as a convenient and rapid approach for epidemiological investigation,and tcpS can be a potential candidate gene for the development of PCR-based Salmonella identification.The serotype distribution of Salmonella tcpS gene was analyzed by bioinformatic approach.The specificity and sensitivity of the PCR method were determined based on 27 different Salmonella serovars and 10 non-Salmonella strains.The PCR method was applied to clinical Salmonella isolates from one pig farm (48 isolates),one chicken farm (22 isolates) and one cattle farm (11 isolates) from Jiangsu Province.In silico analysis showed that tcpS existed only in Salmonella Enteritidis,Pullorum/Gallinarum,and Dublin.The developed PCR method had potent specificity and sensitivity,and could screen the three specific Salmonella serovars accurately.The coincidence rate of the clinical sample detection was up to 100％.The tcpS-based PCR detection method could screen Salmonella Enteritidis,Pullorum/Gallinarum,and Dublin accurately,and could be an assistant method to the traditional serotyping method.Furthermore,the novel tcpS gene can be a potent gene candidate for the development of PCR method for the identification of Salmonella serovars.
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A tcpS-based PCR method was established to simultaneously screen Salmonella enterica serovars Enteritidis,Pullorum/Gallinarum,and Dublin.The developed PCR method provides laboratorial support as a convenient and rapid approach for epidemiological investigation,and tcpS can be a potential candidate gene for the development of PCR-based Salmonella identification.The serotype distribution of Salmonella tcpS gene was analyzed by bioinformatic approach.The specificity and sensitivity of the PCR method were determined based on 27 different Salmonella serovars and 10 non-Salmonella strains.The PCR method was applied to clinical Salmonella isolates from one pig farm (48 isolates),one chicken farm (22 isolates) and one cattle farm (11 isolates) from Jiangsu Province.In silico analysis showed that tcpS existed only in Salmonella Enteritidis,Pullorum/Gallinarum,and Dublin.The developed PCR method had potent specificity and sensitivity,and could screen the three specific Salmonella serovars accurately.The coincidence rate of the clinical sample detection was up to 100％.The tcpS-based PCR detection method could screen Salmonella Enteritidis,Pullorum/Gallinarum,and Dublin accurately,and could be an assistant method to the traditional serotyping method.Furthermore,the novel tcpS gene can be a potent gene candidate for the development of PCR method for the identification of Salmonella serovars.
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Objective: To analyze the clinical characteristics of young patients with acute myocardial infarction (AMI), discuss the key points of health education in young patients with AMI, enhance the understanding of patients, and improve the prognosis of AMI in the young patients. Methods: hTe patients were chosen in XiangyaHospital from September 2012 to September 2013. We consulted the medical records, analyzed the clinical characteristics and results of coronary angiogram in young patients (age≤45), and compared with old patients (age≥60). Results: There were 69 young patients with AMI, about 14.2% of all the patients with AMI. Of the 69 young patients, 59 were male (85.5%) and 10 were female (14.5%). Compared with the old patients, the percentages of smoking, drinking, hyperlipidemia and overweight were much higher;the percentages of hypertension and diabetes were much lower in young patients. The coronary angiogram showed that the constituent ratios of insigniifcant disease and single-vessel disease inthe young patients were higher than those in the old patients; the constituent ratios of double-vessel disease and triple-vessel disease in the young patients were lower than those in the old patients. Conclusion: The clinical characteristics of young patients withAMI are different from the old patients.Health education should be conducted in the youth, and new diet and lifestyle should be advocated.
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The undifferentiated form of nasopharyngeal carcinoma (NPC) is the most common malignant head and neck cancer in South China, especially in Cantonese populations. However, few NPC cell lines have been established from the patients in this region. In this study, we established a new NPC cell line, termed SUNE2, from a Cantonese patient with undifferentiated NPC. This cell line had extremely low concentrations of Epstein-Barr virus (EBV) DNA in long-term culture and expressed low levels of latent membrane protein 1 (LMP1), latent membrane protein 2A (LMP2A), BamH1-A right frame 1 (BARF1), EBV-encoded RNA-1 (EBER1), and EBV-encoded RNA-2 (EBER2) in early passages. SUNE2 cells also showed much stronger transforming ability than 5-8F cells in colony formation assays and anchorage-independent growth assays in soft agar, and they only need 2 weeks to form tumors in nude mice. In summary, the SUNE2 cell line is a new in vitro model that can be used for further research on the mechanisms underlying the occurrence and development of NPC.
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Adult , Animals , Female , Humans , Mice , Asian People , Cell Line, Tumor , Cell Transformation, Neoplastic , Colony-Forming Units Assay , DNA, Viral , Metabolism , Herpesvirus 4, Human , Genetics , Mice, Inbred BALB C , Mice, Nude , Nasopharyngeal Neoplasms , Genetics , Metabolism , Pathology , Virology , Neoplasm Transplantation , RNA, Viral , Metabolism , Viral Matrix Proteins , Metabolism , Viral Proteins , MetabolismABSTRACT
Objective To construct human apolipoprotein O (apolipoprotein O, ApoO) expression vector and obtain recombinant fusion protein thioredoxin (Trx)-ApoO by pET prokaryotic expression system. Methods The ApoO gene fragment from the human liver cDNA library was amplified by PCR. The resulting product was cloned into pET-32a(+) vector and sequenced. The confirmed cDNA was cloned into plasmid E.coli DH10B and then transformed into E.coli BL 21 (DE3) where it was induced to express protein by isopropyl β-D-1-thiogalactopyranoside (IPTG).The fusion protein was purified by Ni-NTA resin. Results The ApoO gene was cloned by PCR and a 519 bp DNA fragment was shown on the agarose electrophoresis. The cloned gene was sequenced and demonstrated to have the same sequence as that of human ApoO gene in GenBank which justified a successful construction of recombinant plasmid. ApoO cDNA gene fragment was induced by IPTG, and a 34 kD recombinant fusion protein Trx-ApoO was tested on sodium dodecyl sulfate polyacrylamide (SDS-PAGE). Conclusion Human ApoO gene is successfully cloned and its recombinant fusion protein Trx-ApoO is expressed.
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The aim of this study was to establish an efficient method for expansion in vitro of natural killer (NK) cells highly purified from human peripheral blood. The CD3-CD56+CD16+ NK cells purified by the negative sorting method of MACS (magnetic microbeads activated cells sorting) were expanded with the different combinations of IL-2, SCF, IL-15 in SCGM (stem cell growth medium) supplemented with 10% human AB serum for 18 days. Cultures were fed with fresh medium and cytokines every 3 days. The sum of cells was counted for evaluating the efficiency of expansion. Then the purity of the CD3-CD56+CD16+ NK cells were determined by flow cytometry and the cytotoxicity to K562 targets was detected by CCK-8 assay in the end. Furthermore, the same way was used to explore the relationship between the efficiency of expansion, cytotoxicity to K562 targets of NK cells and the dose of IL-2. The results showed that after peripheral blood mononuclear cells (PBMNC) were purified by the negative sorting method of MACS, the purity of CD3-CD56+CD16+ NK cells increased from (12.70±2.66)% to (93.03±1.72)%. The CD3-CD56+CD16+ NK cells purified by MACS were expanded with the different combinations of IL-2, SCF, IL-15 in SCGM supplemented with 10% human AB serum for 18 days. The expanding multiple of IL-2/IL-15/SCF group was significantly higher than other groups (p<0.05). The purity of NK cells in the groups with cytokines was not significantly lower than that before expansion (p>0.05). The cytotoxicity of the groups with cytokines was significantly higher than that before expansion. Especially, the cytotoxicity (%) of NK cells in IL-2/IL-15 group and IL-2/IL-15/SCF group was more than 90%. The expanding multiples of low-dose group, medium-dose group and high-dose group were significantly higher than that of zero-dose group (p<0.05), but no significant difference was found between themselves (p>0.05). The cytotoxicity of the groups with IL-2 was significantly higher than that before expansion. Cytotoxicity to K562 cells in high-dose group was significantly higher than that in others (p<0.05); there was no significant difference between low-dose group and medium-dose group (p>0.05). It is concluded that cytokines in the 4 groups were efficient for expansion and the cytotoxicity of highly purified NK cells in vitro. IL-2/SCF/IL-15 combination is the most efficient one among different combinations, and enhanced significantly the cytotoxicity of NK cells against K562 targets. The efficiency of expansion and the cytotoxicity in vitro of NK cells are not related with the dose of IL-2, when IL-2<1,000 U/ml. It is indicated that IL-2 of high-dose (≥1,000 U/ml) may enhance the cytotoxicity of NK cells in vitro more efficiently.
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Humans , CD3 Complex , Allergy and Immunology , CD56 Antigen , Allergy and Immunology , Cell Culture Techniques , Methods , Cell Separation , Methods , Cells, Cultured , Immunophenotyping , Interleukin-2 , Pharmacology , K562 Cells , Killer Cells, Natural , Cell Biology , Allergy and Immunology , Receptors, IgG , Allergy and ImmunologyABSTRACT
BACKGROUND: The connection between Natural killer(NK)-cells and allogeneic bone marrow transplantation(allo-BMT)has aroused increasing attention.OBJECTIVE: To explore the effect of NK cells on graft rejection,hematopoietic and immune reconstitution in mouse undergoing allo-BMT.METHODS: Lethally and nonlethally irradiated BALB/c(H-2d)mice were transplanted with C57BL/6(H-2b)bone marrow plus donor peripheral T cells and/or NK cells.RESULTS AND CONCLUSION: Compared with lethally irradiated and allo-BMT group without infusion of NK cells,the survival rate in lethally irradiated and allo-BMT group with infusion of NK cells significantly enhanced; leukocytes count,expression level of CD19+and CD34+cell count recovered rapidly; expression level of H-2b*cell obviously increased.Expression level of CD34"cell in the group with infusion of NK cells was obviously lower than that of the group without infusion of NK cells at 28 days after transplantation,but there was no significant difference between the 2 groups at 60 days(P > 0.05).In nonlethally irradiated and allo-BMT group without NK cell infusion,expression level of H-2b*cell significantly decreased at 30 days after transplantation,and reduced to before transplantation level at 60 days; while expression of H-2b+cell yet could be detected with more than 80% at 60 days after transplantation in group infused with high and low concentration of NK cells.In alIo-BMT mice,alloreactive NK cell inhibits graft rejection,enhances engraftment,promotes the reconstitution of hematopoiesis and immunity,and increases survival rates.
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Objective To set up the optimized conditions,the amplification of eord blood CDCD+34 cells in vitro were analyzed by comparing the conditions such as different feeder-layers, stimulating-factors or purity/contents of those cells. Methods The cord blood CDCD+34 cells proliferation was analyzed by the methods of MTT, cell counting, and flow eytometer. The amplification and clone-forming ability of cord blood CDCD+34 cells were detected under optimized condition. Resulits The growth rates of cord blood CD+34 cell under optimized conditions(10 times) were significantly higher than that of the control(2.8 times) (P ＜0.01), and the cloneforming ability of cord blood CDCD+34 cells under optimized conditions(CFU-C 36.67±6.11) were also better than that of the control(CFU-C 16.33±1.53) (P ＜0.01). Conclusion The cord blood CD+34 cells proliferation can be promoted in the co-cultured system, and the character of the stem cells were kept well in that system.
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OBJECTIVE@#To express and purify the extra cellular full-length human apolipoprotein M(ApoM).@*METHODS@#The ApoM gene fragment was amplified from the human liver cDNA library by PCR. The resulting product was cloned into pGEXT vector and sequenced. Then the confirmed canstatin cDNA was cloned into plasmid E.coli JM109 and then transformed into E.coli DL21(DE3) where it was induced to express protein by IPTG.@*RESULTS@#The ApoM gene was cloned by PCR and a 560 bp DNA fragment was shown on the agarose electrophoresis. The cloned gene was sequenced and demonstrated to have the same sequence as that of human ApoM gene in GenBank. Then ApoM cDNA gene fragment was induced by IPTG, and a 24 kD recombinant ApoM protein was tested on SDS-PAGE.@*CONCLUSION@#Human ApoM gene is successfully cloned and its recombinant proteins are expressed.
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Humans , Apolipoproteins , Genetics , Apolipoproteins M , Base Sequence , Cloning, Molecular , DNA, Complementary , Genetics , Escherichia coli , Genetics , Metabolism , Lipocalins , Molecular Sequence Data , Polymerase Chain Reaction , Recombinant Proteins , GeneticsABSTRACT
The aim of this study was explore the effect of natural killer (NK) cells on engraftment and reconstitution of hematopoiesis and immunity in mice undergoing allogeneic bone marrow transplantation (allo-BMT). Lethally and nonlethally irradiated BALB/c (H-2(d)) mice were transplanted with C57BL/6 (H-2(b)) bone marrow plus donor peripheral T cells and/or NK cells. Recipient CD34(+), H-2K(b+), CD3(+) and CD19(+) cells were detected by flow cytometry; peripheral blood leukocytes were counted by auto-cytometry; survival rates, engraftment, hematopoietic and immune recovery of recipients in different transplant groups were then observed. The results indicated that as compared with lethally irradiated and allo-BMT group without infusion of NK cells, the survival rate in lethally irradiated and allo-BMT group with infusion of NK cells significantly enhanced (survival rates at 60 days were 70% and 0.0% respectively); leukocyte count, expression level of CD19(+) cell and CD34(+) cell count recovered rapidly; expression level of H-2K(b+) cells obviously increased [(86.68 +/- 4.45)% vs (4.68 +/- 0.32)%]; expression level of CD3(+) cell at day 28 after transplantation obviously decreased [(33.69 +/- 3.36)% vs (50.40 +/- 5.06)%, p < 0.01], at day 60 there was not significant difference between the 2 groups (p > 0.05). In nonlethally irradiated and allo-BMT group without NK cell infusion, the expression level of H-2K(b+) at day 30 after transplantation significantly decreased, and reduced to level before transplantation at day 60; while expression of H-2K(b+) yet could be detected with > 80% at day 60 after transplantation in group infused with high and low concentration of NK cells. It is concluded that in allo-BMT mice, alloreactive NK cell inhibits graft rejection, enhances engraftment, promotes the reconstitution of hematopoiesis and immunity, and increases survival rates.