Differentiation character of adult mesenchymal stem cells and transfection of MSCs with lentiviral vectors

This study examined the differentiation character and pluripotency of mesenchymal stem cells (MSCs) under different conditions. Adult MSCs were initially isolated from the bone marrow of rats, cultured in vitro and identified by flow cytometry. After MSCs were transferred to osteogenic and adipogenic medium respectively, the morphological characterization of induced cells was observed. The expression of marker genes was detected by RT-PCR analysis. Then MSCs were transfected with lenti- viral vectors pGC-FU-Sox9-EGFP. Enhanced green fluorescence protein (EGFP) expression and trans- fection efficiency were determined by fluorescence microscopy. The results demonstrated that EGFP caused no effect on the multilineage potential of adult MSCs. Sox9 gene expression of high level was maintained stable in the transfected MSCs and induced MSCs to differentiate into chondrocytes. Ag- gracan was positive in chondrogenic lineages and the expression of aggracan and type Ⅱ collagenwas significantly increased during MSCs chondrogenic differentiation. It was concluded that Sox9 gene-modified adult MSCs may be promising candidate cells for further studies on tissue engineering. EGFP facilitates the research on MSCs physiological behavior and application in tissue engineering during differentiation both in vitro and in vivo.     

Small interfering RNA inhibits SARS-CoV nucleocapsid gene expression in cultured cells and mouse muscles

SARS-CoV is a newly identified coronavirus that causes severe acute respiratory syndrome （SARS）. Currently, there is no effective method available for prophylaxis and treatment of SARS-CoV infections. In the present study, the influence of small interfering RNA （siRNA） on SARS-CoV nucleocapsid （N） protein expression was detected in cultured cells and mouse muscles. Four siRNA expression cassettes driven by mouse U6 promoter targeting SARS-CoV N gene were prepared, and their inhibitory effects on expression of N and enhanced green fluorescence protein （EGFP） fusion protein were observed. A candidate siRNA was proved to down-regulate N and EGFP expression actively in a sequence-specific manner. The expression vector of this siRNA was constructed and confirmed to reduce N and EGFP expression efficiently in both cultured cells and adult mouse muscles. Our findings suggest that the siRNA should provide the basis for prophylaxis and therapy of SARS-CoV infection in human.     

Inhibition of RAW264.7 Macrophage Inflammatory Cytokines Release by Small Haparin RNAi Targeting TLR4

In order to construct an expression vector carrying small hairpin (sh) RNA (shRNA) for toll-like receptor 4 mRNA and a reporter gene of enhanced green fluorescence protein (EGFP) and study the inhibition of cytokine release by RAW264.7 cell induced by lipopolysaccharide (LPS) stimulation through transfection and expression of shRNA targeting TLR4 gene via the RNAi mechanism, the reporter gene plasmid pEGFP-C1 (4.7 kb) and psiRNA-hHlneo (2979 bp) were used. The H1 promotor and double Bbs I restrict endoenzyme site were cloned from plasmid psiRNA-hH1neo and reconstructed them into plasmid pEGFP-C1 in the Mlu I restrict endoenzymic site, forming plasmid pEGFP-H1/siRNA, which contained Bbs site and reporter EGFP gene. Then an oligonuclear hairpin sequence targeting TLR4 gene was designed by internet tool and inserted into the plasmid pEGFP-H1/siRNA forming plasmid pEGFP-H1/TLR4-siRNA. After transfection of pEGFP-H1/TLR4-siRNA into RAW264.7 cells, tumor necrosis factor-alpha (TNF-α) release by the cells after stimulation by LPS was detected. The results showed that the constructed pEGFP-H1/TLR4-siRNA carrying hairpin RNA for TLR4 gene and reporter EGFP gene were proven to be right by restriction endonuclease analysis. The expression of EGFP gene was (50.37±8.23) % and after transfection of the plasmid pEGFP-H1/ TLR4-siRNA the level of TNF-αreleased by RAW264.7 cell was down regulated. It was concluded that shRNA targeting TLR4 gene could inhibit the TNF-αrelease by RAW264.7 cells evoked by LPS.     

Small interfering RNA inhibits SARS-CoV nucleocapsid gene expression in cultured cells and mouse muscles

SARS-CoV isa newly identified coronavirus that causes severe acute respiratory syndrome （SARS）. Currently, there is no effective method available for prophylaxis and treatment of SARS-CoVinfections. In the present study, the influence of small interfering RNA （siRNA） on SARS-CoV nucleocapsid （N） protein expression was detected in cultured cells and mouse muscles. Four siRNA expression cassettes driven by mouse U6 promoter targeting SARS-CoV N gene were prepared, and their inhibitory effects on expression of N and enhanced green fluorescence protein （EGFP） fusion protein were observed.     

Effects of Biejia Ruangan Tablet(复方鳖甲软肝片)-Containing Serum on Matrix Metalloproteinase-9 and Tissue Inhibitor of Metalloproteinase-1 Expression in Cultured Renal Interstitial Fibroblasts

Objective:To investigate the effects of Biejia Ruangan Tablet(复方鳖甲软肝片方,BRT)- containing serum on the expression of matrix metalloproteinase(MMP-9) and tissue inhibitor of metalloproteinase(TIMP-1) in cultured renal interstitial fibroblasts.Methods:Different BRT-containing sera were prepared by gastric gavages to rats with the high-dose(7 g/kg),mid-dose(3.5 g/kg),and low-dose(1.75 g/kg)BRT respectively.The expression of extracellular matrix in NRK-49 F cells was induced by treatment with human transforming growth factor-β1(recombined human TGF-β1),and BRT-containing serum.Western blotting and Northern blotting were used to measure type Ⅰ and Ⅲ procollagen,MMP-9,and TIMP-1.Results:The high dose BRT-containing serum could decrease the type Ⅰ and Ⅲ procollagen gene expression which boosted by TGF- β1,at the same time cut down TIMP-1 protein and gene expression which increased by TGF- β1(P0.05).Treatment of cells with recombined human TGF- β 1 had no significant effect on MMP-9 expression and BRTcontaining serum also had no effect on MMP-9 expression.Conclusions:High dose BRT has anti-fibrosis effects in NRK-49 F cells,as indicated by its inhibition of type Ⅰ and Ⅲ procollagen and TIMP-1 expression.     

Labeling embryonic stem cells with enhanced green fluorescent protein on the hypoxanthineguanine phosphoribosyl transferase locus

Objective To label embryonic stem(ES)cells with enhanced green fluorescent protein(EGFP)on the hypoxanthineguanine phosphoribosyl transferase(HPRT) gene locus for the first time to provide a convenient and efficient way for cell tracking and manipulation in the studies of transplantation and stem cell therapy.Methods Homologous fragments were obtained by polymerase chain reaction(PCR),from which the gene targeting vector pHPRT-EGFP was constructed.The linearized vector was introduced into ES cells by electoporation.The g4186tG cell clones were obtained after selection with G418 and 6TG media.The integration patterns of these esistant cell clones were identified with Southern blotting. Results EGFP expressing ES cells on the locus of HPRT were successfully generated.They have normal properties,such as karyotype,viability and differentiation ability.The green fluorescence of EGFP expressing cells was maintained in propagation of the ES cells for more than 30 passages and in differentiated cells.Cultured in suspension,the “green”ES cells aggregated and formed embryoid bodies, retaining the green fluorescence at varying developmental stages.The“green”embryoid bodies could expand and differentiate into various types of cells,exhibiting ubiquitous greem fluorescence.Conclusions This generation of “green”targeted ES cells is described in an efficient protocol for obtaining the homologous fragments by PCR.Introducing the marker gene in the genome of ES cells,we should be able to manipulate them in vitro and use them as vehicles in cell-replacement therapy as well as for other biomedical and research purposes.     

Extracellular matrix synthesis and ultrastructural changes of degenerative disc cells transfected by Ad/CMV-hTGF-β1

Objective To determine whether the synthesis of proteoglycan, collagen and associated ultrastructure are related to the adenovirus-mediated gene transferred to adult degenerative cells.Methods Adenovirus/cytomegalovirus human transforming growth fector-β1 (Ad/CMV-hTGF-β1) was used to transfect degenerative cells. Antonopulos method, Miamine method and transmission electrion microscopy were conducted to study the synthesis of proteoglycan, collagen, and ultrastructure, respectively. Cell cultures were established from the nucleus pulpous and annulus fibrosus tissues, which were taken from surgery.Results Nucleus pulpous and annulus fibrosus cells were efficiently transduced by the adenoviral vector carrying hTGF-β1 gene. The synthesis of proteoglycan and collagen increased compared with the control group （P<0.05). The metabolism of cells was slightly improved. No significant toxic effects were found.Conclusions Expression of hTGF-β1 gene is efficient to accelerates proteoglycan synthesis and thus accelerates the improvement of collagen. The function and structure of degenerative cells are improved. Ad/CMV-hTGF-β1 may be suitable for treating disc degeneration.     

Impact of 4HPR on the expression of E-Cad in human bladder transitional epithelial cancer cells T24

Previous researches showed that the expression level of E-Cad in most infiltrating cancer cells was reduced or negative. This study explored whether 4HPR restrained the infiltration of bladder cancer cells through regulating the expression of E-Cad. The infiltrating bladder cancer cells T24 were cultured, and then treated by a proper dosage of drug. Their viability was a determined by MTT method. Western blotting and RT-PCR were adopted to detect the changes of E-Cad gene expression at both pro-tein and mRNA levels. Moreover, immunofluorescent staining and confocal fluorescence microscopy were employed for the observation of the expression of E-Cad. The result showed that, at both mRNA and protein levels, the expression level of E-Cad in T24 cells treated by 4HPR was significantly higher than that of control group, while the β-Cat expression was also relocated from the cell nucleus to cyto-plasm. Our findings suggested that the regulatory function of 4HPR on infiltration of bladder cancer cells T24 is at least partly achieved by regulating the expression of E-Cad.     

Combined expression of CTGF and tissue inhibitor of metalloprotease-1 promotes synthesis of proteoglycan and collagen type II in rhesus monkey lumbar intervertebral disc cells in vitro

Background Low back pain has emerged as a widespread disease often caused by intervertebral disc degeneration.This study aimed to establish an in vitro cell culture model of rhesus monkey lumbar intervertebral discs and to investigate the effect of combined connective tissue growth factor （CTGF） and tissue inhibitor of metalloprotease-1（TIMP-1） expression mediated by adeno-associated virus （AAV） on collagen type Ⅱ and proteoglycan levels.The purpose of these investigations was to explore potential methods for relieving the degeneration of lumbar intervertebral disc cells.Methods Rhesus monkey lumbar intervertebral disc nucleus pulposus cells （NPCs） were isolated by enzyme digestion,cultured, and transduced with rAAV2-CTGF-IRES-TIMP-1, rAAV2-CTGF, or rAAV2-TIMP-1 at a multiplicity of infection （MOl） of 106.The expression of collagen type Ⅱ and proteoglycan was measured using RT-PCR and Western blotting.The synthetic rate of proteoglycan was measured using 35S incorporation.Results Rhesus monkey lumbar intervertebral disc NPCs were transduced with rAAV2-CTGF-IRES-TIMP-1,rAAV2-CTGF, and rAAV2-TIMP-1 and the transduced genes were expressed and detected.Compared to the control,CTGF promoted the synthesis of collagen type Ⅱ and proteoglycan.TIMP-1 showed an enhancing effect on the expression of proteoglycan but no effect on collagen type Ⅱ.Expression of both genes in rhesus monkey lumbar intervertebral disc NPCs significantly enhances the synthesis of proteoglycan and collagen type Ⅱ.Conclusions Single gene transduction of CTGF or TIMP-1 can enhanced synthesis of proteoglycan.CTGF expression can also enhance collagen type Ⅱ protein synthesis.Combined transduction of both CTGF and TIMP1 can significantly promote the expression of proteoglycan and collagen type Ⅱ to levels greater than transduction of a single gene alone.Our study provides a good basis for multi-gene therapy to treat lumbar intervertebral disc degeneration.     

Experimental study on MyoD gene induced differentiation of bone marrow mesenchymal stem cells into myoblasts in vitor

Objective:To explore the possibility of the transfection of MyoD gene induced bone marrow mesenchymal stem cells(MSCs) to differentiate into myoblasts in vitro.Methods:The eukaryotic expression plasmid vector pIRES2-EGRP-MyoD was transfected into MSCs with lipotransfection method,and the positive cells were selected by G418;The expression of MyoD was detected in the transfected MSCs with RT-PCR and the amplified,purified product was identified by sequencing;The reporter gene enhanced green fluorescence protein(EGFP)was observed in the transfected cells under a fluorescent and a laser confocal microscopes;Immunohistochemical methods was used to examine the expressions of MyoD,myogenin,myosin,myoglobin and desmin in the differentiated,cells.The ultrastructure changes of the cells before and after transfection were observed with electron microscopy.Results:The expression of MyoD was detected in the transfected MSCs with RT-PCR and the amplified,purified product was as same in sequence as thwat from Genbank;Green fluorescence was observed in the transfected cells under a fluorescent and a laser confocal microscopes;Immunohistochemical methods indicated that MyoD,myogenin,myosin,myoglobin and desmin were expressed in the transfected cells;The transfected cells showed the morphologcal characteristics of matrue cells with filaments in their cytoplasm.Conclusion:MyoD gene can induce cultured MSCs to successfully differentiate into myoblasts,probably providing an experimental foundation for trauma repair.     

TGF-β1基因转染修饰人退变腰椎间盘髓核细胞

Ｄｅｇｅｎｅｒａｔｉｏｎｏｆｔｈｅｌｕｍｂａｒｄｉｓｃａｆｆｅｃｔｓｔｈｅｍｅｃｈａｎｉｃａｌｐｒｏｐｅｒｔｉｅｓｏｆｔｈｅｓｐｉｎｅａｎｄｉｓｒｅｌａｔｅｄｔｏｃｌｉｎｉｃａｌｌｏｗｂａｃｋｄｉｓｏｒｄｅｒｓ Ｔｈｅｌｉｍｉｔｅｄａｖａｉｌａｂｌｅｔｅｃｈｎｏｌｏｇｙｇｅｎｅｒａｌｌｙｉｓｈｉｇｈｌｙｉｎｖａｓｉｖｅａｎｄｕｓｕａｌｌｙｄｉｓｃｅｃｔｏｍｙｉｓｉｎｖｏｌｖｅｄ ,ｗｈｉｃｈｏｆｔｅｎｃｏｍｐｒｏｍｉｓｅｓｔｈｅｓｔｒｕｃｔｕｒａｌｉｎｔｅｇｒｉｔｙｏｆｔｈｅｓｐｉｎｅ Ｌｏｎｇｔｅ…     

TGF-β1基因转染修饰人退变腰椎间盘髓核细胞

目的　探讨对人退变腰椎间盘细胞以外源性生长因子TGF-β1进行基因修饰的可能性,同时检测相应编码蛋白的表达情况.方法　髓核细胞取自8例因腰椎间盘退变手术病人,在体外进行原代培养,并分作3组.将构建好的分别携带增荧光绿色蛋白EGFP基因（报告基因）和TGF-β1基因的腺病毒载体Ad/CMV-EGFP、Ad/CMV- TGF-β1直接转染细胞,对转染情况进行观察.结果　第一组（Ad/CMV-EGFP转染组）经荧光显微镜下观察有特异性绿色荧光表达,并持续4周,表示外源基因转入成功；第二组（Ad/CMV-TGF-β1转染组）经组化染色证明产生TGF-β1,转染率约30%,而作为对照的第三组则染色基本阴性.结论　采用腺病毒载体可以将TGF-β1基因转染修饰腰椎退变椎间盘细胞,并能表达相应的生长因子TGF-β1,为今后腰椎间盘退变疾病的基因治疗提供了实验基础.     

Ad/CMV- hTGF-beta1 treats rabbit intervertebral discs degeneration in vivo.

To investigate therapeutic efficiency of Ad/CMV- hTGF-beta1 gene for rabbit intervertebral disc degeneration model. 60 Japanese white rabbits were selected to form the 1.5-L6 Anterior-Lateral-Anulus-Fibrosus-Incision-Induced model in order to simulate human intervertebral disc degeneration. 36 rabbits, whose corresponding intervertebral discs were injected with 20 microl (10 x 10(6) pfu) of Ad/CMV- hTGF-beta1 gene, constituted the therapy group, 12 were injected with 20 microl (10 x 10(6) pfu)of Ad/CMV-LacZ gene as comparison group, while 12 were only injected with equivalent capacity of saline for empty comparison group, 3 weeks after injection, examples were taken for investigation of HE staining, MRI, Western Blotting and immunohistochemical research TGF-beta1. Wide distribution of TGF-beta1 was detected by immunohistochemical research in the degenerated annulus fibrosus after injection. Western Blotting research showed significant increase of TGF-beta1 content in intervertebral discs treated with TGF-beta1 gene than comparison groups. MRI signal transformed from low to comparatively high and that intervertebral disc pathological degree improved. Ad/CMV- hTGF-beta1 gene transfection is a potential method to increase TGF-beta1 content and reverse intervertebral disc degeneration.     

基质金属蛋白酶组织抑制剂1基因重组腺病毒载体的构建

目的 为探讨基因治疗逆转或延缓椎间盘退变的可行性,构建及制备人基质金属蛋白酶组织抑制剂1 (tissue inhibitor of metalloproteinase 1,TIMP1) cDNA重组腺病毒载体.方法 以含TIMP1 cDNA序列的质粒为模板,通过PCR方法扩增TIMP1cDNA片段,将TIMP1 cDNA全长定向克隆到Ad5MaxTM腺病毒系统的穿梭质粒pDC316上,构建pDC316-TIMP1穿梭质粒;使用Ad5MaxTM腺病毒包装系统,脂质体介导的穿梭质粒及骨架质粒pBHGlox-E1,3Cre共转染HEK293细胞,同源重组构建含TIMP1 cDNA的重组腺病毒Ad5-TIMP1,通过PCR方法鉴定重组腺病毒Ad5-TIM P1的正确性.通过阴离子柱层析方法纯化重组腺病毒Ad5-TIMP1并测定重组腺病毒感染滴度.结果 经PCR及酶切方法证实pDC316-TIMP1穿梭质粒中存在630 bp大小左右的插入片段,与目的基因TIMP1的cDNA大小一致;经PCR鉴定证实TIMP1 cDNA重组腺病毒载体Ad5-TIMP1构建成功,病毒感染性滴度为1×1010 IU/mL.结论 成功构建TIMP1 cDNA重组腺病毒载体,为应用基因治疗方法逆转或延缓椎间盘退变的研究奠定实验基础.     

腺病毒介导的Sox9基因对椎间盘退变动物模型的疗效

目的:探讨腺病毒介导的Sox9基因对兔腰椎间盘退变模型的疗效。方法:采用新西兰大白兔的纤维环损伤法制作腰椎间盘退变模型,24周后,用注射法将腺病毒介导的Sox9基因注射到退变的椎间盘组织观察退变椎间盘组织的变化,行磁共振扫描拍片观察椎间盘变化情况,同时,取椎间盘用精确定量RT-PCR观察Ⅱ型胶原(Col2 al)mRNA的变化。结果:核磁共振扫描拍片证实椎间盘退变有所恢复,精确定量RT-PCR法证实Sox9基因在转录水平上调了Col2 al。结论:腺病毒介导的Sox9基因对兔椎间盘退变模型有明显治疗作用。     

退变腰椎间盘组织中细胞凋亡及相关基因bcl-2和bax表达的研究

目的:探讨腰椎间盘退变的发病机制。方法:采用原位DNA片段末端标记和免疫组织化学方法,检测40例退变腰椎间盘组织及10例正常腰椎间盘组织细胞凋亡状态及凋亡相关基因bcl-2和bax蛋白的表达。结果:40例退变腰椎间盘组织凋亡指数(AI)均值为70.51,bcl-2蛋白阳性细胞百分比为10.12％,bax蛋白阳性细胞百分比为54.56％。10例正常腰椎间盘组织AI均值为20.02,bcl-2蛋白阳性细胞百分比为53.46％,bax蛋白细胞百分比为10.54％。两组间AI均值、bcl-2蛋白表达、bax蛋白表达差异均有显著性意义(P均<0.05)。bcl-2蛋白表达越弱,bax蛋白表达越强,凋亡细胞数越多。结论:bcl-2和bax蛋白均参与了腰椎间盘组织中细胞凋亡的调节,并在腰椎间盘退行性变发生和发展中发挥着重要作用。     

FasL-844T/C基因多态性与退变腰椎间盘髓核FasL表达的关系

目的 研究FasL-844T/C基因多态性、腰椎MRI和退变的腰椎间盘髓核组织中FasL表达之间的关系.方法 采集105例腰椎间盘突出症患者静脉血和髓核组织;利用基质辅助激光解吸附电离飞行时间质谱分析对FasL-844T/C基因多态性进行了检测;采用S-P法染色检测FasL在髓核组织中的表达.结果MRI研究发现,与CC基因型携带者相比,携带TT基因型的个体腰椎间盘退变的积分显著增高(P=0.003);与CC基因型携带者相比,TT基因型携带者髓核细胞表达FasL的积分有统计学差异(P=0.048),但CT基因型携带者与CC基因型携带者之间髓核细胞表达FasL的积分无统计学差异(P=0.264);腰椎间盘退变积分和椎间盘髓核组织FasL表达无相关性.结论 FasL-844T/C基因多态性与腰椎间盘突出症患者的椎间盘髓核组织FasL表达存在相关性;腰椎间盘退变积分和椎间盘髓核组织FasL表达无相关性.     

Transplantation of gene-modified nucleus pulposus cells reverses rabbit intervertebral disc degeneration

Background  Intervertebral disc degeneration is the main cause of low back pain. The purpose of this study was to explore potential methods for reversing the degeneration of lumbar intervertebral discs by transplantation of gene-modified nucleus pulposus cells into rabbit degenerative lumbar intervertebral discs after transfecting rabbit nucleus pulposus cells with adeno-associated virus 2 (AAV2)-mediated connective tissue growth factor (CTGF) and tissue inhibitor of metalloproteinases 1 (TIMP1) genes in vitro.

Methods  Computer tomography (CT)-guided percutaneous annulus fibrosus injury was performed to build degenerative lumbar intervertebral disc models in 60 New Zealand white rabbits. rAAV2-CTGF-IRES-TIMP1-transfected rabbit nucleus pulposus cells were transplanted into degenerative lumbar intervertebral discs (transplantation group), phosphate-buffered saline (PBS) was injected into degenerative lumbar intervertebral discs (degeneration control group) and normal lumbar intervertebral discs served as a blank control group. After 6, 10 and 14 weeks, the disc height index (DHI) and signal intensity in intervertebral discs were observed by X-ray and magnetic resonance imaging (MRI) analysis. The expression of CTGF and TIMP1 in nucleus pulposus tissue was determined by Western blotting analysis, the synthesis efficiency of proteoglycan was determined by a ³⁵S-sulfate incorporation assay, and the mRNA expression of type II collagen and proteoglycan was detected by RT-PCR.

Results  MRI confirmed that degenerative intervertebral discs appeared two weeks after percutaneous puncture. Transgenic nucleus pulposus cell transplantation could retard the rapid deterioration of the DHI. MRI indicated that degenerative intervertebral discs were relieved in the transplantation group compared with the degeneration control group. The expression of collagen II mRNA and proteoglycan mRNA was significantly higher in the transplantation group and the blank control group compared with the degeneration control group (P <0.05).

Conclusions  CT-guided percutaneous puncture can successfully build rabbit degenerative intervertebral disc models. Both CTGF and TIMP1-transfected cell transplantation helps to maintain disc height, and promotes the biosynthesis of type II collagen and proteoglycan in intervertebral discs, reversing the degeneration of intervertebral discs.