Construction and expression of lentiviral vector of rat β-defensin-2 gene

Objective To construct a lentiviral expression vector of rat β-defensin-2(rBD2)gene, and examine its expression by transfected cultured cells,in order to lay the foundation for experiments in vivo.Methods The totaI RNA of rat epithelial ceils was extracted and rBD2 gene was got with PCR amplification.After double-digested and connected the PCR production and lentiviral vector Lentivirus [containing H1 promoter and green fluorescent protein(GFP)],the lentiviral expression vector of rBD2 gene LV-rBD2 was constructed and confirmed by sequencing.The virus-like particles of LV-rBD2 was packed with lentiviral packaging systems and viral titer was determinated by slow-gradient dilution.Expression of rBD2 was tests with RT-PCR and Western Blot after cultured cells had been infected by LV-rBD2.Results The results of gel electrophoresis and DNA sequencing showed that the rBD2 gene was cloned into the lentiviral vector,the sequence is correct.The lentiviral vector particle packaging was complete.the virus titer was adjusted to 1×105 ifu/μl.RT-PCR and Western-blot showed that rBD-2 gene was expressed.Conclusion The lentiviral expression vector of rBD2 gene LV-rBD2 was constructed successful,and could transfecte cells to express rBD2.     

靶向性反向半胱氨酸天冬氨酸蛋白酶3重组腺病毒诱导肝癌细胞凋亡的实验研究

Objective To investigate the effect and specificity of adenovirus containing r-caspase-3 gene on apoptosis of hepatocellular carcinoma cells. Methods The vector α-fetoprotein enhancer-albumin promotor (pAdTrack-EAFP-PALB) was constructed and the r-caspase-3 gene was subcloned into the vector. The linearized shuttle plasmid was homogenously recombined with AdEasy-1 in BJ5183 cells. The candidate clone was analyzed by restriction endonuclease digestion and sequencing, and then pAdeasy-EAFP-PALB/r-caspase-3 vector was digested with Pac Ⅰ and transfected into AD293 cells for packaging and amplifying.Infection titer and rate of recombinant virus was monitored by green fluorescent protein (GFP) expression.The expression of r-caspase-3 was detected by RT-PCR and Western-blot. The apoptosis of HepG2, 7721, L-02 and MDA- MB- 231 cells was detected by FCM method. Results The sequence of shuttle vector pAdTrack-EAFP-PALB/r-caspase-3 was correct after digestion by restriction endonuclease. Vector pAdeasy-EAFP-PALB/r-caspase-3 was identificated by Pac Ⅰ restriction digestion and PCR. The expression of GFP was observed in the transfected AD293 cells. The expression of r-caspase-3 gene was detected in the infected HepG2 cells by RT-PCR and Western-blot. The cells were infected with recombinant r-caspase-3 after 24 hours, and their apoptotic index were as follows: HepG cells, 48.2%; 7721 cells, 17.7%; L-02 cells, 7.3%;M DA- MB- 231 cells, 0%. Conclusion The recombinant of hepatocellular carcinoma - targeting adenovirus containing r- caspase- 3 gene is constructed successfully and can induce the targeted apoptosis of hepatocellular carcinoma cells, which provides the evidences for future research in hepatocellular carcinoma.     

Construction and identification of a human liver specific microRNA eukaryotic expression vector

MiR-122 is one of the non-coding RNAs which showed its effects on the lipo-metablism, virus infection and HCC forming through regulation of liver gene expression. Its eukaryotic expression vector was constructed by using pSuper which was widely applied in the siRNA expression. The precursor of human miR-122 gene was amplified by polymerase chain reaction （PCR） from the human genomic DNA. The positive clones were screened by PCR and restriction enzyme digestion. The new expression vector of miR-122 was named pHsa-m122. PHsa-m122 and its controls were transfected to HepG2 cells. The miR-122 expression activity was evaluated by GFP122i sensor reporter plasmid through fluorescence detection and Western blot. It was shown that the fluorescence intensity of GFP122si and pHsa-m122 co-transfection group was weaker than that of the controls, so the functional activity of expressed miR-122 was detected. When HepG2 cells were co-transfected with HBV1.3 and pHsa-m122 plasmids, the results showed miR-122 may down-regulate the gene expression of HBV. The human liver specific microRNA eukaryotic expression vector of miR-122 was constructed successfully, which may facilitate further study of its function in the development of liver virus infection diseases and HCC. Cellular ＆ Molecular Immunology.     

人骨形成蛋白7重组腺病毒载体的构建及其在牙周膜细胞中的表达

Objective To investigate the expression of adenovirus-transfected human bone morphogenetic protein-7 (BMP-7) in human periodontal ligament cells(PDLCs) and its effect on PDLCs proliferation after transfection. Methods The BMP-7 fragment was amplified by PCR according to the pCMV-SPORT6-BMP-7, and the fragment was cloned into pShuttle-CMV-BMP-7, then it was homogenously recombined with pBHGE3 in 293 cells to obtain adenovirus expression vector containing BMP-7 (Ad-BMP-7). Ad-BMP-7 was identified and the titer of virus was measured after amplification and purification. Ad-RMP-7 transfected human PDLCs in vitro. The BMP-7 protein expression and proliferation of transfected PDLCs were detected by Western blot and MTT assay respectively. Results PCR analysis confirmed that the human BMP-7 gene was successfully inserted into the adenovirus vector. The titer of the recombinant adenovirus was 1.785×1012 pfu/ml. Expression of BMP-7 protein was detected in PDLCs transfected with Ad-BMP-7. The MTT test showed no significant difference between PDLCs transfected with or without Ad-BMP-7. Conclusions Adenovirus expression vector containing BMP-7 can transfect human PDLCs successfully with high expresion of BMP-7 in PDLCs in vitro.     

Expression of TIMP-3 gene by construction of a eukaryotic cell expression vector and its role in reduction of metastasis in a human breast cancer cell line

The present study is aimed at studying the gene for TIMP-3,a mammalian tissue inhibitor,by constructing arecombinant eukaryotic cell vector for gene therapy in human breast cancer.We obtained the TIMP-3 genefrom the human placent by RT-PCR.TIMP-3 gene was subcloned into pcDNA3.1 vetor from pMD18T vectorby means of gene cloning to construct pcDNA3.1 recombinant vector.Human breast cancer cell lineMDA-MB-453 was transfected with pcDNA3.1-TIMP3 recombinant vector using lipofectamine reagent.Thenthe expression of TIMP-3 and the effect on the metastasis of MDA-MB-453 were examined.The correctconstruction of pcDNA-TIMP3 was identified by means of restriction enzyme analysis,PCR amplication andnucleotide sequencing.Western blotting showed that the transfected cells were able to express TIMP-3,indicating that our construction of the pcDNA-TIMP3 eukaryotic expression vector was constructedsuccessfully.Our experiments further indicated that the potential of metastasis was significantly reduced forthe transfected cell line MDA-MB-453.Cellular & Molecular Immunology.2004;1(4):308-310.     

过表达人抗菌肽LL37腺病毒构建及转染阴道上皮细胞

BACKGROUND: LL37, the only antimicrobial peptide of the cathelicidin family identified from the human, not only promotes the proliferation of endothelial cells, but also plays an important role in angiogenesis and re-epithelialization. OBJECTIVE: To construct a recombinant adenovirus overexpressing human antimicrobial peptide LL37 gene, and to detect the expression and secretion of LL37 after transfected into the canine vaginal epithelial cells. METHODS: The cDNA encoding LL37 was amplified by PCR. Recombinant adenovirus expression plasmid encoding LL37 and green fluorescent protein (EGFP) was constructed, and identified using restriction endonuclease technology and DNA sequencing method. Adenovirus particles were generated by cotransfecting the 293-packaging cell line. The adenovirus were collected, amplified and concentrated, and viral titers were determined by end-point dilution assay by applying serial dilutions of the purified viruses to 293 cells. Primary cultured canine vaginal epithelial cells were transfected by the recombinant adenovirus. The transfection efficacy was observed by fluorescence microscope, and the cultured supernatant was collected to determine the expression of LL37 by ELISA method at 1, 2, 3, 5 and 7 days after transfection. RESULTS AND CONCLUSION:The adenovirus vector GV314-LL37 with the titer of 3×109 pfu/mL was successfully constructed and identified by DNA sequencing methods. Canine vaginal epithelial cells were successfully isolated and cultured and grew stably. After transfection, vaginal epithelial cells could express the EGFP and LL37 efficiently in a time-dependent manner detected by fluorescence microscope and ELISA method. The transfection efficacy of EGFP reached to 89% at 72 hours. The level of LL37 in the cell culture supernatant in the transfection group was significantly higher than that in the control group, the highest expression of LL37 was found at 3 days that lasting for 7 days. In conclusion, the recombinant adenovirus overexpressing human antimicrobial peptide LL37 gene is successfully constructed, which can express and secrete LL37 after transfected into canine vaginal epithelial cells, providing a foundation for constructing the tissue-engineered vagina possessing anti-infection and neovascularization. 中国组织工程研究杂志出版内容重点：组织构建;骨细胞;软骨细胞;细胞培养;成纤维细胞;血管内皮细胞;骨质疏松;组织工程     

Construction and prokaryotic expression of the fusion protein Stx2B-intiminC300 of EHEC O157：H7 and its immunoprophylactic potential

To construct and express the fusion protein Stx2B-IntiminC300 of EHEC 0157 :H7, and to further investigate its immunoprophyiactic potential, the gene of Stx2B (stx2b) from EHEC 0157:H7 chromosome was cloned into pMD18-T vector. Thereafter, the amplified gene was cloned into prokaryotic expression plasmid pET-28a ( )-eaeC300, which was constructed previously. The recombinant pasmid pET-28a( )-stx2b-eaeC300 was transformed into E. coli BL21(DE3). After inducement, the protein Stx2B-IntiminC300 was successfully expressed and analyzed with sodium dodecyl sulfate polyacrylamide gel electrophoresis (SDS-PAGE), Western blotting and N-terminal amino acid residual sequencing. To evaluate its immunoprophyiactic potential, it was primarily purified by ion-exchange chromatography and injected into 30 BALB/c mice with Al(OH)3 in the subscapular region. Ten days after the last booster vaccination, 20 mice were attacked with EHEC 0157: H7 lysate and the protective efficacy was observed. In the present study, the gene of Stx2B-IntiminC300 was successfully cloned into pET-28a ( ) vector. The results of SDS-PAGE and Western blotting assay showed that the fusion protein was successfully expressed in the inclusion body form, accounting for 25% of total expression products, and its molecular weight was about 43 kDa. The result of the N-terminal amino acid residual sequencing showed that it was identical to that of the molecular designed. The purity was about 75 % after primary purification. Animal tests revealed that the fusion protein Stx2B-IntiminC300 has elicited high titer of protective antibody relatively. These results demonstrate that the fusion protein Stx2B-IntiminC300 is successfully expressed in prokaryotic expression system and shows certain immunoprophyiactic potential.     

Effect of PKC signalling pathway and aldose reductase on expression of fibronectin induced by transforming growth factor-β1 in human mesangial cells

Objective To study the effect of PKC signalling pathway and aldose reductase (AR) on the expression of fibronectin (FN) induced by transforming growth factor-β1 (TGF-β1). Methods Human mesangial cells (HMCs) were cultured and transfected with pcDNA3-AR, and subject to AR gene silencing with small interfering RNA (siRNA) and then the cell was treated with recombinant human TGF-β1. The AR mRNA expression in the HMCs was examined using real time RT-PCR and protein expression of AR and FN was detected by Western blotting. Results The cultured HMC treated with TGF-$l showed increased expression of AR and FN,the normal HMC showed not reduced expression of FN after incubation with single inhibitors of AR. Pre-incubation of cells with inhibitors of AR and PKC, then the different groups of cells were treated with TGF-$l ,and the induction effect on FN expression was suppressed (34%) in HMC. HMCs transfected with AR showed a strong protein expression of FN, which was increased by 3. 6-fold after treatment with TGF-pl (P <0. 05) , and the induction effect on FN expression was suppressed by G(O)6983 (42%) in HMCs (P < 0. 05) . The HMC with AR gene knock-down by siRNA showed a decreased expression of AR and 90% decrease of FN protein in HMCs(P <0. 01) , and TGF-β1-induced up-regulation of FN was significantly suppressed by siRNA (12%) in HMCs (P <0. 01). Conclusions AR is capable of regulating FN expression only in the presence of TGF-β1, and this reaction is possibly accomplished through the activation of PKC signalling pathway.     

Effect of PKC signalling pathway and aldose reductase on expression of fibronectin induced by transforming growth factor-β1 in human mesangial cells

Objective To study the effect of PKC signalling pathway and aldose reductase (AR) on the expression of fibronectin (FN) induced by transforming growth factor-β1 (TGF-β1). Methods Human mesangial cells (HMCs) were cultured and transfected with pcDNA3-AR, and subject to AR gene silencing with small interfering RNA (siRNA) and then the cell was treated with recombinant human TGF-β1. The AR mRNA expression in the HMCs was examined using real time RT-PCR and protein expression of AR and FN was detected by Western blotting. Results The cultured HMC treated with TGF-$l showed increased expression of AR and FN,the normal HMC showed not reduced expression of FN after incubation with single inhibitors of AR. Pre-incubation of cells with inhibitors of AR and PKC, then the different groups of cells were treated with TGF-$l ,and the induction effect on FN expression was suppressed (34%) in HMC. HMCs transfected with AR showed a strong protein expression of FN, which was increased by 3. 6-fold after treatment with TGF-pl (P <0. 05) , and the induction effect on FN expression was suppressed by G(O)6983 (42%) in HMCs (P < 0. 05) . The HMC with AR gene knock-down by siRNA showed a decreased expression of AR and 90% decrease of FN protein in HMCs(P <0. 01) , and TGF-β1-induced up-regulation of FN was significantly suppressed by siRNA (12%) in HMCs (P <0. 01). Conclusions AR is capable of regulating FN expression only in the presence of TGF-β1, and this reaction is possibly accomplished through the activation of PKC signalling pathway.     

大鼠C-sis基因克隆及其真核表达载体的构建

BACKGROUND: C-sis proto-oncogene can promote tissue repair by inducing cell proliferation and inhibiting cell apoptosis. Therefore, C-sis may play a positive role in the repair of damaged liver tissue and the treatment of fulminant hepatic failure. OBJECTIVE: To construct pcDNA3.1/C-sis eukaryotic expression vector and detect its expression in BRL cells (the normal liver cells of rats) and rat liver cells in vivo. METHODS: The full-length coding sequence of C-sis gene was cloned through real time-PCR. pcDNA3.1/C-sis eukaryotic expression vector was constructed and sequenced, followed by transfected into BRL cells using liposome and injected into the rat liver via tail vein. Finally, its expression in BRL cells and rat liver cells in vivo was identified by fluorescence quantitative PCR and western blotting. RESULTS AND CONCLUSION: (1) The full length of encoding region of C-sis gene was successfully cloned. Sequencing proved that pcDNA3. 1/C-sis recombinant eukaryotic expression vector was constructed successfully. (2) The expression of C-sis was increased after transfected into BRL cells and rat liver. (3) These results provide basis for the subsequent study of the effect of C-sis gene on fulminant hepatic failure in rats. 中国组织工程研究杂志出版内容重点：肾移植;肝移植;移植;心脏移植;组织移植;皮肤移植;皮瓣移植;血管移植;器官移植;组织工程     

Gene transfer to dendritic cells induced a protective immunity against melanoma

Lentiviral vectors have shown promises for efficient gene transfer to dividing as well as nondividing cells. In this study, we explored lentiviral vector-mediated, the entire mTRP-2 gene transfer and expression in dendritic cells （DCs）. Adoptive transfer of DCs-expressing mTRP-2 （DC-HR＇CmT2） into C57BL/6 mouse was also assessed. Dendritic cells were harvested from bone marrow and functional DCs were proved by allogeneic mixed lymphocyte reaction. Lentiviral vectors were produced by transient transfection of 293T cells. Transduction of DCs was proved by marker gene expression and PCR and RT-PCR amplification. Implantation of the transduced DCs, depletion of immune cells as well as the survival of the mice after tumour challenge were investigated. High efficiency of gene transfer into mature DCs was achieved. The high level expression of the functional antigen （TRP-2） and induction of protective immunity by adoptive transfer of TRP-2 gene modified DCs were demonstrated. In vivo study showed a complete protection of mice from further melanoma cell challenge. In comparison, only 83% of mice survived when mTRP-2 peptide-pulsed DCs were administered, suggesting the generation of specific protection. Together, these results demonstrated the usefulness of this gene transfer to DC approach for immunotherapy of cancer and indicated that using tumour associated antigens （TAAs） for gene transfer may be potentially beneficial for the therapy of melanoma.     

联合应用实时定量RT-PCR和激光显微切割检测单个肝细胞RNA的表达（英文）

AIM: The manner in which a cell responds to and influences its environment is ultimately determined by the genes that are expressed. To better understand cellular functions, the isolation of single cells and subsequent quantification of the expressed genes is essential. METHODS: Normal liver tissue was obtained from operation, snap-frozen in liquid nitrogen and sectioned in crystat. Individual hepatocytes were microdissected. RNA was extracted, then reverse transcribed and amplified using real-time quantitative polymerase chain reaction (PCR). RESULTS: Single hepatocytes were dissected by laser beam and catapulted to the microcentrifuge cap which was put above the slide. In this way, cells were collected, RNA was extracted, reverse transcribed to cDNA and used for analysis of RNA expression by real-time quantitative PCR. The amplification results showed that quantitation of the RNA inside the cell was compatible with the number of cells. CONCLUSION: The expression of RNA in single cells can be quantitated successfully by using laser microdissection and real-time PCR. These techniques provide an opportunity to monitor in vivo gene expression levels in single hepatocytes.     

构建肿瘤坏死因子α刺激基因6慢病毒表达及干扰载体及对人瘢痕疙瘩成纤维细胞增殖与凋亡的影响

BACKGROUND: Current research has shown that tumor necrosis factor α stimulated gene 6 (TSG-6) has anti-inflammatory effect, and the scar formation can be inhibited by local injection of TSG-6 protein at the early stage of trauma. However, the mechanism of this effect is still unclear. OBJECTIVE:To construct the lentiviral expression vector and shRNA vector for human TSG-6, with stable overexpression, transfection and interference, and to explore the effect of TSG-6 on proliferation and apoptosis of keloid fibroblast cell lines.  METHODS:Human keloid fibroblast cells were isolated from the keloid’s tissue by enzyme digestion and identified by immunocytochemistry assay. Lentiviral vectors pLVX-puro-TSG-6 and pLVX-shRNA1-TSG-6 were constructed and transfected into human keloid fibroblast, exclusively. Expression levels of TSG-6 mRNA and protein were detected by RT-PCR and western blot assay. MTT assay and flow cytometry were used to estimate the cell proliferation and apoptosis in each group after transfection. In addition, expression of Bcl-2, p53 and active-caspase-3 were detected by western blot assay in each group. RESULTS AND CONCLUSION: (1) Human keloid fibroblasts were separated successfully. Under the light microscope, cells were spindle. Immunohistochemical staining for vimentin was performed in the fifth passage of cells, with the positive rate of 100%. Cells were negative for cytokeratin. (2) Recombinant lentiviral vectors and stably transfected cell lines were successfully established. TSG-6 gene expression was altered apparently. Compared with the control group, cell proliferation was delayed and apoptotic rate was noticeably increased in TSG-6 gene overexpression group. Cell proliferation increased and apoptotic rate decreased in the TSG-6 gene intervention group (P < 0.05). (3) Western blot assay results demonstrated that Bcl-2 expression reduced, P53 and Active-caspase-3 expression significantly increased in the TSG-6 gene overexpression group (P < 0.05). (4) These finding showed that TSG-6 could inhibit proliferation and induce apoptosis in keloid fibroblasts. Its mechanism may be associated with the down-regulation of Bcl-2 protein expression, up-regulation of P53 protein expression and increased Caspase-3 activity. 中国组织工程研究杂志出版内容重点：组织构建;骨细胞;软骨细胞;细胞培养;成纤维细胞;血管内皮细胞;骨质疏松;组织工程     

IL-24基因修饰的树突状细胞促进CIK细胞对肺癌细胞的杀伤作用

Objective To study the antitumor effect and mechanism of co-cultured cytokine-induced killer(CIK) cells and autologous DC modified with IL-24 gene on A549 cells in vitro. Methods DC and CIK cells were prepared routinely from human peripheral blood mononuclear cells(PBMC). Recombinant adenovirus vector pAdEasy-1-pTrack-CMV-IL-24 was extracted from DH5α, it was lineared with Pac I and transfected into A293 cells, and then the IL-24 recombined adenovirus(Ad-IL-24) was obtained. Ad-IL-24 was used to infect DC. The cells obtained were named DC-IL-24. RT-PCR and ELISA were used to evaluate the expression of IL-24 gene in transfected DC. The phenotypes change of DC were identified by flow cytometry analysis, the concen-tration of IL-12 and TNF-α in supernatant of DC were determined by EIJSA. The ability of CIK producing per-forin was measured by homolysis method. FCM was used to determine the cytotoxicity of cocultured CIK cells and autologous DC modified with IL-24 gene to A549 cells. Results We obtained the high titre of Ad-IL-24.IL-24 gene was transfered into DC successfully via Ad-IL-24. The green fluorescence was observed on DC by fluorescence microscope. The expression rate of CD80, CD83, HI.A-DR, CD40, CXCR4 on DC-IL-24 was sig-nificantly increased compared with that of the control group. DC-IL-24 produced markedly higher levels of IL-12 and TNF-α as compared with DC. DC-IL-24 can enhance the ability of CIK cells producing perforin. On com-parison with non-transfected DC co-cultured with CIK cells, transfected DC co-cultured with CIK cells had a sig-nificantly higher lytic activity against A549 cells. Conclusion IL-24 gene modification can enhance the anti-tu-moral immunity of DC. The mechanism of which might be related to the increased secretion of IL-12 and TNF-α, up-regulation expression of co-stimulatory molecules and MHC Ⅱ class molecules on DC, promoting the acti-vation and maturation of DC, and then enhancing CIK cells to generate specific anti-tumoral immunity.     

人脐动脉平滑肌细胞培养及转染TFPI基因的实验研究

Objective The aim of the present study is to investigate the effect of tissue factor pathway inhibitor (TFPI) gene on cell cycle of human vascular smooth muscle cells.Methods Human vascular smooth muscle cells were separated from human umbilical artery and identified by immunohistochemical staining.The cells were transfected with various amount of pIRES-TFPI plasmid (1,2,and 3 μg/ml,respectively)and the TFPI expression in the cells were analyzed by RT-PCR.MTT assay was employed to detect the effect of TFPI gene on the proliferation of human vascular smooth muscle cells.Results The proliferation of vascular smooth muscle cells was inhibited in pIRES-TFPI group 5 and 7 days after gene transfection when compared with that of pIRES 1-neo transfection group.Conclusion The overexpression of TFPI gene in human umbilical artery vascular smooth muscle cells may contribute to the suppression of the proliferation of cells by gene transfection.     

寡聚蛋白Trim34α对NF-κB报告基因的负向调节作用

目的 测Trim34α对TAB2诱导的NF-κB报告基因活化的影响.结果 经鉴定成功构建Trim34α真核表达载体,该载体表达的Trim34α蛋白能相互聚集形成寡聚体(Trim小体);Trim34α可显著抑制TAB2诱导的NF-κB荧光素酶报告基因活化.结论 Trim34α可以在胞内形成寡聚体,Trim34α能显著抑制TAB2诱导的NF-κB报告基因活化.

Abstract：

Objective To investigate the effects of Trim34α on the activation of luciferase reporter gene containing NF-κB promoter induced by adaptor proteins TAB2. Methods The total RNA was isolated from HeLa cells. After amplification with RT-PCR, the target sequences were cloned into 5'-Flag-pcDNA3.1 (+) vector. The recombinant vector was confirmed by restriction enzyme digestion, colony PCR and sequencing. It was transfected into HEK293T cells to detected Trim34α expression by Western blot. Simultaneously, the effects of Trim34α on the NF-κB activation induced by TAB2 were determined by dual-luciferase reporter assay. Results Restriction enzyme digestion, colony PCR and sequencing confirmed the vector was constructed successfully, furthermore it expressed Trim34α protein in HEK293T cells. Moreover, trim34α could form high-molecular-weight oligomeric protein, and here we called it trimsome. Interestingly, dual-luciferase assay showed that Trim34α could effectively block TAB2-induced NF-κB activation. Conclusion Trim34α was involved in negative regulation of TAB2-induced NF-κB activation and could form high-molecular-weight oligomer.     

构建线粒体钙离子摄入蛋白1慢病毒表达载体及在H9C2细胞中的应用

BACKGROUND: Mitochondrial calcium uptake 1 (MICU1) is one of the important molecules to maintain the mitochondrial calcium homeostasis. The regulation of MICU1 to mitochondrial calcium homeostasis may play an important role in diabetic cardiomyopathy, but the underlying mechanism remains unclear. OBJECTIVE:To construct a lentiviral vector carrying MICU1 gene to transfect H9C2 cells, and then to assess MICU1 level in H9C2 cells thereby establishing a platform for researching the occurrence and development of diabetic cardiomyopathy at a cellular level. METHODS: DNA fragments of MICU1 were amplified by PCR, cleaved with Spe I, EcoR I and cloned into the lentiviral vector pRRLsin.CMV.eGFP to construct pRRLsin.CMV.MICU1-eGFP vector. 293T cells were co-transfected with recombined pCMVDR8.91 and pCMV-VSVG to produce pRRLsin.CMV.MICU1-eGFP lentiviral viruses, and then used to infect H9C2 cells. mRNA and protein expressions of MICU1 in the transfected H9C2 cells were evaluated by real-time PCR and western blot assay. Mitochondrial calcium level in Rhod-2-stained H9C2 cells was tested under confocal microscope. RESULTS AND CONCLUSION: The recombinant inducible lentiviral vector containing MICU1 gene was successfully constructed. 293T could express green fluorescent protein with increased MICU1 level after pRRLsin.CMV.MICU1-eGFP transfection. The mRNA and protein expressions of MICU1 in the infected H9C2 group were obviously up-regulated compared with the other groups. MICU1 could remarkably improve the mitochondrial calcium level under Rhod-2 staining. These results show that pRRLsin.CMV.MICU1-eGFP lentiviral viruses are efficient to transfect H9C2 cells, which will be powered to lay a foundation for the immortalized cell line establishment. 中国组织工程研究杂志出版内容重点：组织构建;骨细胞;软骨细胞;细胞培养;成纤维细胞;血管内皮细胞;骨质疏松;组织工程