An Investigation of the Effects of B7-H4 Gene rs10754339 and miR-125a Gene rs12976445 on Cancer Susceptibility

Objective To investigate the effects of the B7-H4 gene rs10754339 and miR-125a gene rs12976445 on cancer susceptibility through a case-control study and meta-analysis.Methods A total of 1,490 cancer patients(lung/gastric/liver/:550/460/480)and 800 controls were recruited in this case-control study.The meta-analysis was performed by pooling the data from previous related studies and the present study.Results The results of this study showed that in the Hubei Han Chinese population,the rs10754339gene was significantly associated with the risk of lung and gastric cancer but not liver cancer,and the rs12976445 gene was significantly associated with the risk of lung cancer but not liver or gastric cancer.The meta-analysis results indicated that rs10754339 and rs12976445 contributed to cancer susceptibility in the Chinese population and also revealed a significant association between rs10754339and breast cancer risk,as well as between rs12976445 and lung cancer risk.Conclusion The B7-H4 gene rs10754339 and miR-125a gene rs12976445 may be the potential genetic markers for cancer susceptibility in the Chinese population,which should be validated in future studies with larger sample sizes in other ethnic populations.

Prediction of Tumor Microenvironment Characteristics and Treatment Response in Lung Squamous Cell Carcinoma by Pseudogene OR7E47P-related Immune Genes

Objective Pseudogenes are initially regarded as nonfunctional genomic sequences,but some pseudogenes regulate tumor initiation and progression by interacting with other genes to modulate their transcriptional activities.Olfactory receptor family 7 subfamily E member 47 pseudogene(OR7E47P)is expressed broadly in lung tissues and has been identified as a positive regulator in the tumor microenvironment(TME)of lung adenocarcinoma(LUAD).This study aimed to elucidate the correlation between OR7E47P and tumor immunity in lung squamous cell carcinoma(LUSC).Methods Clinical and molecular information from The Cancer Genome Atlas(TCGA)LUSC cohort was used to identify OR7E47P-related immune genes(ORIGs)by weighted gene correlation network analysis(WGCNA).Based on the ORIGs,2 OR7E47P clusters were identified using non-negative matrix factorization(NMF)clustering,and the stability of the clustering was tested by an extreme gradient boosting classifier(XGBoost).LASSO-Cox and stepwise regressions were applied to further select prognostic ORIGs and to construct a predictive model(ORPScore)for immunotherapy.The Botling cohorts and 8 immunotherapy cohorts(the Samstein,Braun,Jung,Gide,IMvigor210,Lauss,Van Allen,and Cho cohorts)were included as independent validation cohorts.Results OR7E47P expression was positively correlated with immune cell infiltration and enrichment of immune-related pathways in LUSC.A total of 57 ORIGs were identified to classify the patients into 2 OR7E47P clusters(Cluster 1 and Cluster 2)with distinct immune,mutation,and stromal programs.Compared to Cluster 1,Cluster 2 had more infiltration by immune and stromal cells,lower mutation rates of driver genes,and higher expression of immune-related proteins.The clustering performed well in the internal and 5 external validation cohorts.Based on the 7 ORIGs(HOPX,STX2,WFS,DUSP22,SLFN13,GGCT,and CCSER2),the ORPScore was constructed to predict the prognosis and the treatment response.In addition,the ORPScore was a better prognostic factor and correlated positively with the immunotherapeutic response in cancer patients.The area under the curve values ranged from 0.584 to 0.805 in the 6 independent immunotherapy cohorts.Conclusion Our study suggests a significant correlation between OR7E47P and TME modulation in LUSC.ORIGs can be applied to molecularly stratify patients,and the ORPScore may serve as a biomarker for clinical decision-making regarding individualized prognostication and immunotherapy.

Codominance Functional Marker of Bacterial Blight Resistance Gene Xa7 in Rice

[Objectives]A codominance functional marker of the broad-spectrum bacterial blight resistance gene,Xa7,of rice was identified for accurate detection,generation tracking,and differentiation between homozygous and hemizygous genotypes of the gene.[Methods]A potential functional marker containing four primers was designed using Premier 5 software and based on the differences on the sequences of Xa7,xa7,and allele-free genomes.The molecular distinctness of the marker in different materials was verified by PCR.Three crossbreed lines of Xa7 and their parents were inoculated with seven bacterial blight strains at the booting stage to examine the affected agronomic traits at maturation.[Results]The homozygous R084 of Xa7 could be amplified into a 91 bp band and the Nip free of allele with a 153 bp band,while the heterozygote Nip/R084,91 bp and 153 bp bands.The candidate codominance marker,Xa7fun,amplified fragments that matched the predicted target bands.No 91 bp fragment was amplified from 18 germplasms of varied types,indicating a lack of Xa7 in them.Whereas Ry1,Ry2 and Ry3 had a 91 bp band,suggesting the inclusion of homozygous Xa7.Under an elevated temperature,Huazhan responded to the seven bacterial blight pathogens as highly susceptible(HS),intermediate susceptible(MS),or susceptible(S);R084 to six of the seven pathogens(HNA1-4,FuJ,GDA2,GD1358,PX086,and YN24)as highly resistant(HR),intermediate resistant(MR)or resistant(R);Ry-1 to five pathogens(GDA2,HNA1-4,FuJ,GD1358,and YN24)as HR or MR;Ry-2 to five pathogens(GDA2,GD1358,HNA1-4,PXO86,and YN24)as HR or R;and Ry-3 to 6 pathogens(HNA1-4,FuJ,GDA2,GD1358,PXO86,and YN24)as HR or MR.Therefore,the infiltration of Xa7 in the improved crossbred lines RY-1,RY-2,and RY-3 significantly accentuated the blight resistance of Huazhan.[Conclusions]Homozygous or hemizygous Xa7 could be accurately differentiated by the currently identified codominance functional marker Xa7 fun.The Xa7 introgression did not significantly alter the critical agronomic traits in the hybridization from generation to generation and could be safely applied in breeding rice varieties with bacterial blight resistance.

Identification of immune feature genes and intercellular profiles in diabetic cardiomyopathy

BACKGROUND Diabetic cardiomyopathy(DCM)is a multifaceted cardiovascular disorder in which immune dysregulation plays a pivotal role.The immunological molecular mechanisms underlying DCM are poorly understood.AIM To examine the immunological molecular mechanisms of DCM and construct diagnostic and prognostic models of DCM based on immune feature genes(IFGs).METHODS Weighted gene co-expression network analysis along with machine learning methods were employed to pinpoint IFGs within bulk RNA sequencing(RNA-seq)datasets.Single-sample gene set enrichment analysis(ssGSEA)facilitated the analysis of immune cell infiltration.Diagnostic and prognostic models for these IFGs were developed and assessed in a validation cohort.Gene expression in the DCM cell model was confirmed through real time-quantitative polymerase chain reaction and western blotting techniques.Additionally,single-cell RNA-seq data provided deeper insights into cellular profiles and interactions.RESULTS The overlap between 69 differentially expressed genes in the DCM-associated module and 2483 immune genes yielded 7 differentially expressed immune-related genes.Four IFGs showed good diagnostic and prognostic values in the validation cohort:Proenkephalin(Penk)and retinol binding protein 7(Rbp7),which were highly expressed,and glucagon receptor and inhibin subunit alpha,which were expressed at low levels in DCM patients(all area under the curves>0.9).SsGSEA revealed that IFG-related immune cell infiltration primarily involved type 2 T helper cells.High expression of Penk(P<0.0001)and Rbp7(P=0.001)was detected in cardiomyocytes and interstitial cells and further confirmed in a DCM cell model in vitro.Intercellular events and communication analysis revealed abnormal cellular phenotype transformation and signaling communication in DCM,especially between mesenchymal cells and macrophages.CONCLUSION The present study identified Penk and Rbp7 as potential DCM biomarkers,and aberrant mesenchymal-immune cell phenotype communication may be an important aspect of DCM pathogenesis.

Roles and regulation of bone morphogenetic protein-7 in kidney development and diseases

The gene encoding bone morphogenetic protein-7(BMP7) is expressed in the developing kidney in embryos and also in the mature organ in adults. During kidney development, expression of BMP7 is essential to determine the final number of nephrons in and proper size of the organ. The secreted BMP7 acts on the nephron progenitor cells to exert its dual functions: To maintain and expand the progenitor population and to provide them with competence to respond to differentiation cues, each relying on distinct signaling pathways. Intriguingly, in the adult organ, BMP7 has been implicated in protection against and regeneration from injury. Exogenous administration of recombinant BMP7 to animal models of kidney diseases has shown promising effects in counteracting inflammation, apoptosis and fibrosis evoked upon injury. Although the expression pattern of BMP7 has been well described, the mechanisms by which it is regulated have remained elusive and the processes by which the secretion sites of BMP7 impinge upon its functions in kidney development and diseases have not yet been assessed. Understanding the regulatory mechanisms will pave the way towards gaining better insight into the roles of BMP7, and to achieving desired control of the gene expression as a therapeutic strategy for kidney diseases.

Anti-gastric cancer active immunity induced by FasL/B7-1 gene-modified tumor cells

AIM: To study the activation of cytotoxic T lymphocytes (CTLs) against gastric cancer cells induced by FasL/B7-1 (FB-11) gene-modified tumor cells, and to explore whether co-expression of FasL and B7-1 in SGC-7901 tumor cells could initiate synergistic antitumor effect. METHODS: FasL and B7-1 genes were transfected into human SGC-7901 gastric cancer cells with adenovirus vectors. The positive clones were selected by G418. FasL and B7-1 genes were detected by flow cytometry and RT-PCR. Abdominal infiltrating lymphocytes and sensitized spleen cells were obtained from mice that were immunized with SGC-7901/FB-11 or wild type SGC-7901 cells intraperitoneally, and cytotoxicity of these CTLs against tumor cells was determined by MTT assay. RESULTS: Flow cytometry and RT-PCR showed that FasL and B7-1 genes were highly expressed. FasL and B7-1 transfected cancer cells had a high apoptosis index. DNA laddering suggested that FasL and B7-1 genes induced gastric cancer cell apoptosis. FasL+/B7-1+SGC-7901 cells (SGC-7901/FB-11) were inoculated subcutaneously in the dorsal skin of C57BL/6 mice and then decreased their tumorigenicity greatly (z = 2.15-46.10, P＜0.01). SGC- 7901/FB-11 cell-sensitized mice obtained protective immune activity against the rechallenge of wild type SGC 7901 cells (z = 2.06-44.30, P＜0.05). The cytotoxicity of CTLs induced by SGC-7901/FB-11 cells against SGC-7901 was significantly higher than that of CTLs activated by wild-type SGC-7901 cells (84.1±2.4% vs30.5±2.3%,P＜0.05).CONCLUSION: FasL and B7-1 genes can effectively promote the activity of CTLs against gastric cancer cells. FasL/B7-1 molecules play an important role in CTL cytotoxicity.

Let-7a gene knockdown protects against cerebral ischemia/reperfusion injury

The micro RNA（mi RNA） let-7 was one of the first mi RNAs to be discovered, and is highly conserved and widely expressed among species. let-7 expression increases in brain tissue after cerebral ischemia/reperfusion injury; however, no studies have reported let-7 effects on nerve injury after cerebral ischemia/reperfusion injury. To investigate the effects of let-7 gene knockdown on cerebral ischemia/reperfusion injury, we established a rat model of cerebral ischemia/reperfusion injury. Quantitative reverse transcription-polymerase chain reaction demonstrated that 12 hours after cerebral ischemia/reperfusion injury, let-7 expression was up-regulated, peaked at 24 hours, and was still higher than that in control rats after 72 hours. Let-7 gene knockdown in rats suppressed microglial activation and inflammatory factor release, reduced neuronal apoptosis and infarct volume in brain tissue after cerebral ischemia/reperfusion injury. Western blot assays and luciferase assays revealed that mitogen-activated protein kinase phosphatase-1（MKP1） is a direct target of let-7. Let-7 enhanced phosphorylated p38 mitogen-activated protein kinase（MAPK） and c-Jun N-terminal kinase（JNK） expression by down-regulating MKP1. These findings suggest that knockdown of let-7 inhibited the activation of p38 MAPK and JNK signaling pathways by up-regulating MKP1 expression, reduced apoptosis and the inflammatory reaction, and exerted a neuroprotective effect following cerebral ischemia/reperfusion injury.

Sequence Analysis of Type III Effector tccP and tccP2 Genes in Escherichia coli O157:H7 from Chinese Water-chestnut

[Objective] This study aimed to analyze the type III effector tccP and tccP2 genes in Escherichia coli O157：H7 from Chinese water-chestnut. [Method] Gene-specific and locus-specific primers were utilized to amplify tccP/tccP2 and their flanking regions for sequence analysis. [Result] E. coli O157：H7 CWN11 harbored intact tccP and tccP2 genes, however, the number of proline-rich repeats in tccP gene was only one that probably resulted in biological incapability, whereas, the tccP2 gene consisted of five and half proline-rich repeats and could encode functional protein. [Conclusion] Here, we reported the first sequence of tccP gene that consisted of only one proline-rich repeat and tccP2 was assumed to play a crucial role in colonization and subsequent signaling cascades.

Expression of Porcine Reproductive and Respiratory Syndrome Virus ORF7 Gene and Purification and Immunological Activity Analysis of the Recombinant Protein

[Objective] The aim of this study was to realize efficient expression of the porcine reproductive and respiratory syndrome virus （PRRSV） ORF7 gene in genetic engineering bacteria and analYze the immunological activity of the recombinant protein after purification. [ Method] The constructed recombinant expression vector pET-ORF7 was transformed into Escherichia co1BL21 （DE3） and induced by IPTG under the optimal condition. After analysis of SDS-PAGE and Western Blot, the expression products were purified by Ni-NTA His · Bind Resin chrom- atographic column under denaturing condition and renatured by gradient dialysis. Subsequently, the immunological activity of the renatured recombinant protein was detected by Westem Blot and indirect ELISA. [ Result] The recombinant plasmid pET-ORF7 expressed in E. coli successfully, and the fusion protein was in the form of inclusion body. By SDS-PAGE detection, the molecular weight of the expression protein was approximate 33 kD, according with the expectation. Analysis by Bandscan software showed that the expressed fusion protein was about 50% of total bacterial protein of BL21 （DE3）. Wastem Blot and indirect ELISA detection showed that the renatured protein could react with PRRSV positive serum specifically, indicating its good immunological activity. [ Conclusion] This study lays a foundation for the preparation of PRRSV monoclonal antibody and diagnostic kit.

Construction of Eukaryotic Expression Vector Containing B7-1/GFP Gene and Its Expression in Osteosarcoma Cell Line

Objective： To construct eukaryotic expression vector containing B7-1/GFP geneand study its expression in osteosarcoma cell line LM8. Methods： By using gene cloning technique, eukaxyotic expression vector pEGFP-C1 was used to construct the murine B7-1 recombinant plasmid （pEGFP-C1/B7）. Recombinant plasmid was transfected into LM8 cells with liposome and was confirmed by restriction endonuclease digestion and DNA sequencing. The expression of the fusion protein was detected using fluorescence microscope and Western blot analysis. Results： The recombinant eukaryotic expression plasmid pEGFP-C1/B7 was successfully constructed, which was confirmed by DNA sequencing, RT-PGR and restriction enzymes analysis. The green fluorescent protein could be detected in the transfected LM8 with fluorescence microscope. The expected B7-1 and green fluorescent protein （GFP） fusion protein was detected by RT-PCR and Western blot. Conclusion： The eukaryotic expression vector containing B7-1/GFP gene was constructed successfully, and it could be expressed in LM8 after transfection.

Construction of Adeno-associated Virus System for Human Bone Morphogenetic Protein 7 Gene

To construct the recombinant adeno-associated virus （rAAV） vector with human bone morphogenetic protein 7 （BMP7） and observe the BMP7 mRNA expression in vitro, BMP7 CDS sequence was cloned into expression plasmid pAAV-MCS of AAV Helper Free System. The recombinant plasmid was identified with enzyme digestion and sequencing. The recombinant plasmid, pAAV-RC, pHelper were co-transfected into AAV-293 cells according to the calcium phosphate-based protocol. The viral stock was collected by 4 rounds of freeze/thaw. After purified and concentrated, the recombinant virus titer was determined by dot-blot assay. HEK293 cells were transfected with the recombinant virus at different MOI, and the expression of BMP7 mRNA was detected by RT-PCR. The results showed rAAV-BMP7 was constructed and packaged successfully. The physical particle titer was 2.5×10^11 vector genomes/mL. There was different expression level of BMP7 mRNA after transfecton. These data suggested that recombinant AAV mediated a stable expression of hBMP7 mRNA in 293 cells. The AAV production method may pave the way of an effective strategy for the jaw bone defection around dental implants.

Genetic association study of P2x7 A1513C(rs 3751143) polymorphism and susceptibility to pulmonary tuberculosis: A meta-analysis based on the findings of 11 case-control studies

Objective:To summarize the precise association between pulmonary tuberculosis(PTB) and P2x7 A1513 C gene polymorphism.Methods:PubMed and Google Scholar web-databases were searched for the studies reporting the association of P2x7 A1513 C polymorphism and PTB risk.A meta-analysis was performed for the selected case-control studies and pooled odds ratios(ORs) and 95%confidence intervals(95%CIs) were calculated for all the genetic models.Results:Eleven studies comprising 2 678 controls and 2 113 PTB cases were included in this meta-analysis.We observed overall no significant risk in all the five genetic models.When stratified population by the ethnicity,Caucasian population failed to show any risk of PTB in all the genetics models.In Asian ethnicity,variant allele(C vs.A:P=0.001;QR=1.375,95%CI=1.159-1.632) and heterozygous genotype(AC vs.AA:P=0.001;OR=1.570,95%CI=1.269-1.944) demonstrated significant increased risk of PTB.Likewise,recessive genetic model(CC+AC vs.AA:P=0.001;OR=1.540,95%CI= 1.255-1.890) also demonstrated increased risk of PTB in Asians.Conclusions:Our meta-analysis did not suggest the association of P2x7 A1513 C polymorphism with PTB risk in overall or separately in Caucasian population.However,it plays a significant risk factor for predisposing PTB in Asians.Future larger sample and expression studies are needed to validate this association.

Recombinant adenovirus vector-mediated human MDA-7 gene transfection suppresses hepatocellular carcinoma growth in a mouse xenograft model

Hepatocellular carcinoma is one of the most common tumors in the world. The purpose of the present study was to investigate the inhibitory effects of adenoviral transduction of human melanoma differentiation-associated gene-7 （MDA-7） gene on hepatocellular carcinoma, so as to provide a theoretical basis for gene therapy of the disease. The human MDA-7 gene was cloned into replication-defective adenovirus specific to HepG2 cells us- ing recombinant virus technology. RT-PCR and Western blotting assays were used to determine the expression of human MDA-7 mRNA and MDA-7 protein in HepG2 cells in vitro. Induction of apoptosis by overexpression of the human MDA-7 gene was determined by flow cytometry. In-vivo efficacy of adenoviral delivery of the hu- man MDA-7 gene was assessed in nude mice beating HepG2 cell lines in vivo by determining inhibition of tumor growth, VEGF and CD34 expression, and microvascular density （MVD）. The results showed that AdGFP/MDA- 7 induced apoptosis of HepG2 cells in vitro and significantly inhibited tumor growth in vivo （P 〈 0.05）. The intra- tumoral MVD decreased significantly in the treated tumors （P 〈 0.05）. We conclude the recombination adenovirus AdGFP/MDA-7 can effectively express biologically active human MDA-7, which leads to inhibition of hepatocel- lular carcinoma growth.

Transfection of Articular Chondrocytes with rhBMP7 Gene and Its Expression

In order to investigate the possibility of expression of exogenous gene in transduced articular chondrocytes, plasmid pcDNA3 rhBMP7 was delivered to cultured chondrocytes. Through immunohistochemical staining and RT PCR assay, the expression of rhBMP7 gene was detected. And the bioactivity of transgene expression product was detected through MTT assay as well. It was confirmed that exogenous gene could be expressed efficiently in transduced chondrocytes and the transgene expression product had obvious bioactivity. The present study provided a theoretical basis for gene therapy on the problems of articular cartilge.

Transforming Activity of a Novel Mutant of HPV16 E6E7 Fusion Gene

An optimized recombinant HPV16 E6E7 fusion gene (HPV16 ofE6E7) was constructed according to codon usage for mammalian cell expression, and a mutant of HPV16 ofE6E7 fusion gene (HPV16 omfE6E7) was generated by site-directed mutagenesis at L57G, C113R for the E6 protein and C24G, E26G for the E7 protein for HPV16 ofE6E7 [patent pending (CN 101100672)]. The HPV16 omfE6E7 gene constructed in this work not only lost the transformation capability to NIH 3T3 cells and tumorigenicity in SCID mice, but also maintained very good stability and antigenicity. These results suggests that the HPV16 omfE6E7 gene should undergo further study for application as a safe antigen-specific therapeutic vaccine for HPV16-associated tumors.

Molecular Cloning and Sequence Analysis of FullLength cDNA Encoding Human Bone Morphogenetic Protein—7

Objective:To clone the full-length human bane morphogenetic protein-7 (BMP-7 ) gene and analyse its sequence, to aid in investigation of its function and structure. Methods : Total RNA was isolated from Chinese fetal kidney by the acid gmnidinium thiocyanate phenol-chloroform method. Two overlapping segments of human BMP- 1 cDNA were obtained by reverse transcription (RT)-PCR. Following application, the two segments were ligated to each other and subcloned into POEM-T easy vector to form PEGM-T easy/hBMP-7 recombinant plasmid. Sanger dideoxy chain-termination method was used to sequence the cDNA. Results. There was 750 bp fragment obtained RT-PCR using #2 primer from 5' end of BMP-7 gene (PCR by using # 2 and # 1) ,and 540 bp fragment from 3' end was generated by KT-PCR using # 4 primer (PCR using # 3 and # 4). Full-length cDNA encoding BMP-7 was obtained by religation of two segments. When compared with hBMP-7 sequence in Gene bank (XM30619) ,our full-length BMP-7 cDNA has a G instead of a T at nucleotide 862. This change results in valine substituting for phenylalanine in the protein. Conclusion. This is the first time that BMP-7 cDNA was successfully cloned from Chinese fetal kidney. BMP-7 cDNA plays an important role in healing injuries of the osteo-articular system. This makes BMP-7 is an attractive target far various clinical applications.

Experimental Study on the Antitumor Effect of Mouse B7-1 Gene

Summary: Mouse B7 1 cDNA was cloned by RT PCR from BALB/C mouse splenic cells and inserted into pcDNA3 to construct an eukaryotic expression vector. This constructor was named pCD mB7 1, in which the B7 1 cDNA was identified to be consistent with the data from other researchers. pCD mB7 1 plasmid was transfected into B16(F0) cells, and effective expression of mB7 1 in these tumor cells could be detected till the 6th month by RT PCR and RNA hybridization. Specific cytotoxity assay of lymphocytes was conducted after culturing with tumor cells and the results demonstrated that B16 cells transfected with B7 1 gene were more effective than B16 wt and B16 neo in inducing specific cytotoxity of lymphocytes against B16 wt cells. It is suggested that expression of B7 1 gene in tumor cells could enhance the immunogenicity and induce the effective antitumor immunity.

STUDY OF ENHANCED IMMUNOGENECITY OF B7-1 GENE TRANSFECTED HUMAN HELA CELL LINE

This work was supposed by CMB (No. 96—635) This is one of papers of the special issue on gene therapy research (Chin J Cancer Res Vol. 9 No. 4 December, 1997). Although cervical carcinoma cells may express the human papillomavirus protein E6 and E7, they fail to induce an effective specific cytotoxic T lymphocyte response. Recent studies suggest that expression of CD 80 (B7 1) on tumor cells is effective to induce antitumor immune responses. 1,2 In our study, CD 80 gene was transfected into human Hela cell line with a CD 80 expression plasmid (B7 1 +pcDNA 3) by electroporation, then the immunogenecity of the modified Hela cell was tested in TLMC (tumor lymphocyte mixed culture) system. Thymidine lymphocyte proliferation assays showed that the response of human peripheral blood lymphocytes (PBLS) to CD 80 positive Hela cells demonstrated a substantial increase in cell proliferation compared to the response to control cells. Cocultivation of allogeneic PBLs with CD 80 positive tumor cells for three days can induce an increased secretion of IL 2. Our results demonstrate an immunostimulatory effect of CD 80 expression on cervical cancer cells, which provides a basis for the development of a therapeutic tumor vaccine.

The Analysis of Human Papillomavirus Type 16 E6/E7 Genetic Variability in Jingjiang, Jiangsu Province, China

Human papillomavirus 16 (HPV-16) is a major high-risk type causing cervical cancer. The E6 and E7 genes in HPV-16 are the major virulent genes in HPV, and we wanted to investigate the polymorphism of E6 and E7 genes of HPV-16 originated from Jingjiang, Jiangsu province. In research, HPV-16 sample was collected, and the E6 and E7 genes fragments were amplified by PCR assay. The sequences of E6 and E7 genes were used for phylogenetic analysis by Mega 5.0 software. By comparison, the major mutations of E6 gene were T178G (41.67%) and G658A (17%), as well as C58G, T61A and G188C (14%), T61C and C656A (11%). The mutation of E7 gene was mainly C491A and T935A (23%);G514C, G937C and G519C (11%). The 68 nucleotide site deficiency (69.4%) of E6 and 535 nucleotide site deficiency (23%) of E7 were dominant. In our study, we did not find the relevance between the E6 and E7 genes mutations and pathologic severity. But we think the E6 and E7 genes mutation of HPV-16 might affect vaccine prevention and treatment effect.

EXPRESSION OF rhBMP-7 GENE IN TRANSDUCED BONE MARROW DERIVED STROMAL CELLS

OBJECTIVE: To explore the possibility of expression of exogenous gene in transduced bone marrow derived stromal cells (BMSCs). METHODS: The marker gene, pbLacZ, was transferred into cultured BMSCs and the expression of transduced gene by X-gal staining was examined. Then plasmid pcDNA3-rhBMP7 was delivered to cultured BMSCs. Through immunohistochemical staining and RT-PCR assay, the expression of rhBMP7 gene was detected. RESULTS: The exogenous gene could be expressed efficiently in transduced BMSCs. CONCLUSION: The present study provided a theoretical basis to gene therapy on the problems of bone and cartilage tissue.