人前脑啡肽原基因修饰人骨髓间充质干细胞系的构建

目的 构建人前脑啡肽原(PENK)基因修饰的并可稳定分泌脑啡肽蛋白的人骨髓间充质干细胞系(hMSCs).方法 采用脂质体法将PENK基因逆转录病毒载体质粒(pBABE-PENK)转染至Phoenix-293T细胞,收集病毒上清液感染hMSCs细胞,经过嘌呤霉素筛选得到稳定表达PENK基因的hMSCs细胞株(hMSC-PENK细胞).以转染空载体细胞作为对照,即hMSC-pBABE细胞.采用RT-PCR法检测PENK mRNA的表达,免疫荧光法测定亮氨酸脑啡肽(LEK)的表达,ELISA法测定细胞培养上清液LEK浓度.结果 与hMSCs细胞和hMSC-pBABE细胞比较,hMSC-PENK细胞PENK mRNA和LEK表达上调,细胞培养上清液中LEK浓度升高(P＜0.05或0.01).结论 PENK基因修饰的hMSCs可表达PENK基因并分泌脑啡肽蛋白,成功构建了稳定分泌镇痛物质的细胞系.     

利用Tet-on调控系统建立人肝癌HepG2Tet-on细胞系

目的 构建可以用强力霉素调控表达的人肝癌HepG2Tet-on细胞系,为进一步研究肝癌相关基因功能奠定基础.方法 用脂质体转染法将pWHE146质粒转染到人肝癌HepG2细胞中,用G418筛选出稳定表达细胞克隆;单克隆分别扩增后,瞬时转染pTRE-hyg-luc质粒;强力霉素诱导表达后,检测荧光素酶表达活性,挑选出受强力霉素调控的低背景、高表达的HepG2Tet-on细胞株.结果 成功构建了一株受强力霉素调控的高表达低背景的HepG2Tet-on细胞株(诱导倍数达154.106倍).结论 HepG2Tet-on细胞株可用于外源基因的真核调控高表达,为研究真核基因功能提供一种可靠的细胞株.     

可控性Speedy A基因表达转基因小鼠模型的建立鉴定和表型分析

目的建立基于Tet-On系统诱导表达的Speedy A基因RNA干扰转基因小鼠模型,探讨Tet-On系统在研究精子发生相关基因中的应用,研究Speedy A基因下调对精子发生的影响。方法体外实验筛选下调Speedy A基因表达的有效干扰片段,Gateway技术构建包含有效干扰片段和四环素反应元件(TRE)的质粒(pRP.EX2d-TREshSpdya),受精卵显微注射该质粒建立转基因小鼠并筛选到纯合子,然后与四环素调控的反式激活子(rtTA)转基因小鼠杂交,筛选TRE和rtTA双阳性的小鼠为四环素诱导的Speedy A-RNA干扰转基因小鼠模型,喂食强力霉素(Dox)诱导RNA干扰片段的表达下调Speedy A基因的表达;实时荧光定量PCR法检测模型组小鼠与喂食强力霉素的正常小鼠(对照组)Speedy A的表达差异;HE染色观察睾丸组织形态学变化,TUNEL法检测小鼠睾丸组织的细胞凋亡情况。结果模型组小鼠Speedy A基因的表达量与对照组小鼠的表达量相比下降26%;模型组小鼠睾丸组织发生异常的生精细胞凋亡。结论成功建立了基于Tet-On系统诱导表达的可控性Speedy A-RNA干扰转基因小鼠模型,说明可以基于Tet-On系统在体研究精子发生相关基因的功能;Speedy A基因表达下调可使生精细胞发生异常凋亡。     

EphA1基因可控性双稳转内皮祖细胞系EPCsTet-On-EphA1SiRNA的建立

目的建立可调控EphA1基因表达的内皮祖细胞系EPCsTet-On-EphA1SiRNA。方法将pWHE146质粒转染到内皮祖细胞系中,筛选出稳定表达的细胞克隆;扩增后瞬时转染pTRE-hyg-luc质粒,强力霉素诱导表达后,检测荧光素酶活性,挑选出高表达、低背景的受强力霉素调控的EPCsTet-On细胞株;再将重组质粒pTRE-EphA1SiRNA转染入EPCsTet-On细胞株,筛选出稳定表达细胞克隆EPCsTet-On-EphA1SiRNA;通过强力霉素诱导后,利用RT-PCR和Western blotting法检测EphA1基因mRNA与蛋白的表达。结果成功构建了受强力霉素调控的高表达低背景的EPCsTet-On-EphA1SiRNA细胞株;强力霉素可诱导EPCsTet-On-EphA1SiRNA细胞株中EphA1mRNA表达下调,较之未调控组其差异有统计学意义(P<0.05);强力霉素调控EphA1蛋白表达的能力在一定范围内呈剂量依赖性关系。结论成功建立强力霉素调控EphA1基因表达的大鼠双稳转内皮祖细胞系EPCsTet-On-EphA1SiRNA,为深入研究EphA1基因在内皮祖细胞参与肝癌血管生成过程中的作用提供了有效的实验手段。     

携带可调控人前脑啡肽原基因逆转录病毒载体的构建及鉴定

目的构建四环素调控的含人前脑啡肽原基因(hPPE)逆转录病毒载体。方法用PCR方法扩增目的基因hPPE,定向克隆入逆转录病毒四环素反应质粒(pRevTRE)中,酶切反应、PCR 及DNA测序鉴定重组逆转录病毒载体pRevTRE/hPPE;在脂质体介导下分别将pRevTRE/hPPE和逆转录病毒四环素调节质粒(pRevTet-on)转入包装细胞PT67,经相应的抗生素稳定筛选得到抗性细胞克隆。RT-PCR鉴定得到阳性产病毒细胞系PT67/Tet-on和PT67/TREhPPE。用NIH3T3细胞测定病毒滴度。结果经限制性酶切分析、RT-PCR及DNA序列分析证实pRevTRE/hPPE中含有hPPE基因。转染有pRevTet-on的FT67细胞病毒滴度为2.6×105 CFU/ml,转染有pRevTRE/hPPE的PT67细胞病毒滴度为3.2×105 CFU/ml。结论成功构建了含有受四环素调控hPPE基因的逆转录病毒载体,建立了能产较高滴度逆转录病毒的包装细胞系,为可调控细胞移植镇痛的研究奠定了实验基础。     

Tet-on基因表达系统反应质粒P~(TRE-HIF-1α)的构建和表达鉴定

目的克隆和构建携带人低氧诱导因子-1α（HIF-1α）的Tet-on基因表达系统反应质粒P^TRE-HIF-1α。方法以缺氧的肝癌细胞株HepG2总RNA为模板，进行RT-巢式PCR，获得HIF-1α的cDNA，克隆人Tet-on基因表达系统反应质粒P^TRE2hyg，酶切重组子鉴定。将构建好的P^TRE-HIF-1α用脂质体法转入HepG2^Tet-on细胞，在强力霉素的作用下，用RT-PCR及Western blot法鉴定重组质粒的表达。结果扩增出HIF-1α的eDNA测序结果与Genbank记载完全一致，并成功克隆人P^TRE2hyg，将反应质粒P^TRE-HIF-1α转入HepG2^Tet-on细胞，可以完整有效表达HIF-1α且受强力霉素的调控。结论成功克隆和构建携带HIF-1α基因的Tet-on基因表达系统反应质粒P^TRE-HIF-1α，并证明其表达能在HepG2^Tet-on细胞中受强力霉素调控。     

Smad7对高糖介导大鼠肾小管上皮细胞转分化和胶原I合成的影响

目的探讨Smad7对高糖介导肾小管上皮细胞转分化和胶原（Col）I合成的影响。方法体外培养转染Smad7的大鼠近端肾小管上皮细胞株（NRK52E细胞），分为强力霉素（Dox）诱导组和未加强力霉素的对照组。前者予Dox诱导24h后，给予高糖刺激；后者不加Dox诱导。采用免疫细胞化学方法检测磷酸化（p）-Smad2／3核转位情况；RT-PCR检测Smad7的表达；Western印迹方法检测不同时间点Smad7、α-SMA、E-钙黏蛋白（cadherin）和Col I蛋白的表达水平。结果成功构建了Dox调控的可上调Smad7表达的NRK52E细胞。NRK52E细胞在基础状态下可表达低水平p-Smad2／3（16．1％），与未加Dox的对照组比较，Dox诱导组可显著抑制高糖刺激的NRK52E细胞TGF-β受体调控信号蛋白Smad2／3的活化和核转位（30.3％比58．5％，P〈0．01）。上调表达Smad7可显著抑制高糖介导的NRK52E细胞α-SMA和Col I蛋白的表达；逆转高糖介导的NRK52E细胞E-cadherin蛋白的下调表达。结论基因转染上调表达Smad7可通过TGF-β受体调控信号蛋白Smad2／3的活化和核转位而阻抑高糖介导的肾小管上皮细胞转分化及细胞外基质的合成。     

四环素调控系统修饰永生化大鼠星形胶质细胞株的构建及鉴定

目的构建四环素及其衍生物强力霉素诱导表达目的基因的永生化大鼠星形胶质细胞株。方法用脂质体法将逆转录病毒载体四环素调控系统中的质粒pRevTet-On转染病毒包装细胞 PT67,经筛选培养后获得病毒载体RevTet-On,采用RT-PCR进行鉴定,并应用NIH313细胞测定病毒滴度。将RevTet-On感染永生化大鼠星形胶质细胞,用有限稀释法挑选阳性细胞单克隆后扩大培养,每个克隆瞬时转染含萤光素酶报告基因的质粒pRevTRE-Lue,加入强力霉素48h后分别检测其萤光素酶活性值,挑选出强力霉素诱导表达高、背景表达低的细胞株并检测其诱导表达的时效、量效关系。结果 RT-PCR结果显示逆转录病毒包装成功,病毒最高滴度为7．4×105CFU／ml。RevTet-On转染永生化大鼠星形胶质细胞后挑选出48个单克隆,瞬时转染pRevTER-Luc后筛选出1株高表达低背景细胞株, 其诱导表达值为876．1 RLU,背景表达值为42．5 RLU,诱导倍数为20．6。该细胞株在加入诱导因子强力霉素1 h后目的基因即开始表达,在24 h时达到高峰,在强力霉素浓度100-2 000 ng/ml的范围内, 其诱导表达活性与药物浓度呈剂量依赖性。结论含四环素调控系统的永生化大鼠星形胶质细胞株诱导活性可靠,可用于调控表达外源基因的研究。     

人前脑啡肽基因修饰人胚胎肾细胞的构建

目的 构建人前脑啡肽基因(hPPE)修饰的人胚胎肾细胞(HEK293细胞).方法 重组质粒pcDNA3.1(+)/hPPE经限制性内切酶HindⅢ和Not Ⅰ进行双酶切获得hPPE基因,运用基因重组技术将hPPE基因与表达载体同源重组,转染293T细胞进行慢病毒包装、扩增、纯化,测定病毒滴度,再将重组的慢病毒载体转染HEK293细胞.用Western blot法检测hPPE基因在HEK293细胞中的表达.结果 重组慢病毒载体阳性克隆测序结果和基因库的hPPE基因序列完全一致.含有hPPE基因的慢病毒载体,滴度为2.07×108TU/ml.转染慢病毒载体后的HEK293细胞,在荧光显微镜下未见到GFP荧光.转染Ubc-GFP-L.V.空病毒载体的HEK293细胞,可见到较强的荧光.Western blot法检测到经慢病毒载体转染后的HEK293细胞中hPPE基因表达呈阳性.结论 成功构建了hPPE基因修饰的HEK293细胞,使hPPE基因可在HEK293细胞中稳定表达.     

重组人前脑啡肽原基因腺相关病毒载体的构建

目的 构建重组人前脑啡肽原基因(hPPE)腺相关病毒载体(AAV)。方法 构建质粒骨架上含有新霉素抗性基因表达盒的重组腺相关病毒(rAAV)载体质粒pSNAV-hPPE并酶切鉴定,转染金黄地鼠胚胎肾细胞(BHK 21),后用G418筛选培养形成稳定携带rAAV载体的混合细胞株BHK／SH1;再用HSV1-rc／△UI2感染、包装此细胞株并收获病毒载体rAAV-hPPE,并行DNA探针点杂交测定病毒滴度。结果 酶切鉴定pSNAV-hPPE重组成功,病毒滴度为2.5×1012v.g.／ml。结论 获得的rAAV-hPPE滴度高,感染性好,可以用于转基因镇痛的实验研究。     

Construction and identification of immortalized rat astrocyte cell line expressing enkephalin

Objective ： To provide a sound cell source for further ex-vivo gene therapy for chronic pain, we attempt to develop an immortalized rat astrocyte cell line that expresses enkephalin regulated by doxycycline. Methods. Retrovirus infection method was employed to develop an immortalized rat astrocyte cell line that could express enkephalin regulated by doxycycline. The hPPE gene expression level of immoralized astroyte cells （IAC）/ hPPE was detected by RT-PCR, indirect immunofluorescence staining and radioimmunoassay. Results. IAC carrying Tet-on system transfected with preproenkephalin gene could secrete enkephalin that was regulated by doxycycline in a dose-dependent manner and hPPE gene activation could be repeated in on-off-on cycles through administration or removal of doxycycline. Conclusion： An immortalized rat astrocyte cell line that secrete enkephalin under the control of doxycycline is established successfully, which provides a research basis for transgenic cell transplantation for analgesia.     

Genetically engineered human mesenchymal stem cells produce met-enkephalin at augmented higher levels in vitro

We have reported that transplantation of adrenal medullary chromaffin cells that release endogenous opioid peptides into pain modulatory regions in the CNS produce significant antinociceptive effects in patients with terminal cancer pain. However, the usefulness of this procedure is minimal because the availability of human adrenal tissue is very limited. Alternative xenogeneic materials, such as porcine and bovine adrenal chromaffin cells present problems of immune rejection and possible pathogenic contamination. In an attempt to develop opioid peptide-producing cells of autologous origin, we have transfected human mesenchymal stem cells (hMeSCs) with a mammalian expression vector containing a fusion gene of green fluorescent protein (GFP) and human preproenkephalin (hPPE), a precursor protein for enkephalin opioid peptides. Enkephalins are major neurotransmitters that play an important role in analgesia by activating peripheral opioid receptors. Following the establishment of stable transfection of hMeSCs, the expressions of hPPE and GFP were confirmed and the production of methionine enkephalin (Met-enkephalin) was significantly increased compared to control naive hMeSCs (p < 0.05). Our in vitro data demonstrated that genetically engineered hMeSCs with transfected hPPE gene can constitutively produce opioid peptide Met-enkephalin at an augmented high level. hMeSCs are relatively easy to isolate from a patient's bone marrow aspirates and expand in culture by repeated passages. Autologous hMeSCs would not require immunosuppression when transplanted back into the same patient. Through targeted gene manipulation such as hPPE gene transfection, this may offer a virtually unlimited safe cell supply for the treatment of opioid-sensitive pain in humans.     

Immunological purification of rat precartilaginous stem cells and construction of the immortalized cell strain

Precartilaginous stem cells (PCSC) are adult stem cells that control limb growth of animals and can differentiate directionally. In a previous study, PCSC was reported to begin differentiating at the fifth passage; therefore, sufficient uni-phenotype PCSC cannot be harvested from primary cell culture. The purpose of this study was to examine whether simian virus 40 large T antigen gene (SV40Tag) could induce rat PCSC to immortalize. Immunomagnetic separation was used to isolate PCSC labeled with fibroblast growth factor receptor-3 (FGFR-3). Plasmid pCMVSV40T/PUR containing SV40Tag was transfected into PCSC by liposome transfection method. One anti-puromycin cell clone was obtained, which was confirmed as FGFR-3 positive, and expanded to immortalized cell strain. Results from RT-PCR and immunocytochemistry demonstrated that SV40Tag was highly expressed at both mRNA and protein levels after stable transfection. The cells transfected with SV40Tag were expanded to immortalized cell strain, which could maintain its characteristics for 30 passages, named immortalized precartilaginous stem cells (IPCSC). IPCSC were short fusiform or triangular cells with two or three short axons. Immunocytochemistry results of FGFR-3 and Collagen II demonstrated that IPCSC retained the characteristics of PCSC and the high proliferation capability of IPCSC was confirmed by methyl thiazolyl tetrazolium assay. Therefore, we concluded that rat precartilaginous stem cells were purified and immortalized precartilaginous stem cell strain was established. It may provide a stable cell resource for basic research and cell transplantation therapies.     

猿肾病毒40介导的人前软骨干细胞永生化的实验研究

目的 构建猿肾病毒40大T抗原基因(SV40Tag)介导的永生化人前软骨干细胞株,为下一步基因打靶研究其分化分子机制提供稳定的细胞来源. 方法采用脂质体介导的基因转染技术将含有SVd0Tag的质粒pCMVSV40T/PUR转染人前软骨干细胞(PSCs),经嘌呤霉素筛选,阳性克隆扩大培养并连续传代.用免疫组化、RT-PCR、Southern印迹杂交法对转染细胞进行鉴定,并检测SV40Tag在转染细胞中的表达及其与基因组的整合情况. 结果 筛选获得的阳性克隆扩大培养,命名为永生化前软骨干细胞(IPSCs),能连续传代培养,细胞生长迅速.免疫组化和RT-PCR证实IPSCs成纤维生长因子受体-3阳性,并可检测到SV40Tag mRNA及其蛋白的表达.Southern印迹杂交显示IPSCs基因组中存在SV40Tag cDNA. 结论 成功构建了SV40Tag介导的永生化人前软骨干细胞株.     

单纯疱疹病毒Ⅰ型扩增子载体转移脑啡肽基因在神经细胞的表达

目的：研究采用单纯疱疹病毒I型扩增子载体将脑啡肽基因导入神经细胞，观察载体lacZ基因和外源脑啡肽基因的表达。方法：实验共分4组；A组为对照组，未加病毒悬液的神经细胞；B组，加病毒悬液的神经细胞培养2d；C组，加病毒悬液的神经细胞培养3d；D组，加病毒悬液的神经细胞培养至第10d。留部基用放射免疫法检测外源性脑啡肽基因的表达。用X-gal原位检测lacZ基因表达反映扩增子病毒的存在和滴度。结果：L－脑啡肽放射免疫检测证实转基因神经细胞L－脑啡肽含量较对照组明显升高（P＜0．05），X-gal染色证实加入假病毒悬液的神经细胞有30％以上的细胞蓝染.结论：HSV－1扩增子假病毒能将外源脑啡肽基因导入神经细胞，并使之有效表达。     

Curcumin attenuates the expression and secretion of RANTES after spinal cord injury in vivo and lipopolysaccharide-induced astrocyte reactivation in vitro

Curcumin has been proposed for treatment of various neuroinflammatory and neurodegenerative conditions, including post-traumatic inflammation during acute spinal cord injury (SCI). In this study, we examined whether curcumin anti-inflammation involves regulation of astrocyte reactivation, with special focus on the injury-induced RANTES (regulated on expression normal T-cell expressed and secreted) from astrocytes in acute SCI. Male Sprague-Dawley (SD) rats were subjected to impact injury of the spinal cord followed by treatment with curcumin (40 mg/kg i.p.). RANTES and inducible nitric oxide synthase expression as well as RANTES-positive astrocytes were all induced by injury accompanied by the elevation of lipid peroxidation, and attenuated by the curcumin treatment. In primary cultured rat astrocytes challenged with lipopolysaccharide (LPS) to mimic astrocyte reactivation following SCI, LPS induces robust increase of RANTES expression and the effect was also reduced by 1 μM curcumin treatment. Furthermore, cortical neurons cultured with astrocyte conditioned medium (ACM) conditioned with both LPS and curcumin (LPS-curcumin/ACM), which characteristically exhibited decreased RANTES expression when compared with ACM from astrocytes treated with LPS alone (LPS/ACM), showed higher level of cell viability and lower level of cell death as assessed by 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide (MTT) reduction activity assay and lactate dehydrogenase release assay, respectively. Knockdown of RANTES expression by siRNA (siRANTES) shows reduced RANTES expression and release from LPS-reactivated astrocytes, and ACM obtained from this condition (LPS-siRANTES/ACM) becomes less cytotoxic as compared with the LPS-ACM. Therefore, curcumin reduction of robust RANTES production in reactivated astrocytes both in vitro and in vivo may contribute to its neuroprotection and potential application in SCI.     

Tet-on基因表达系统调控HIF-1α表达对肝癌细胞增殖的影响

目的 探讨HIF-1α表达对肝癌细胞增殖和细胞周期的影响.方法 利用Tet-on基因表达系统调控肝癌细胞系HepG2中HIF-1α的表达,检测细胞周期和细胞增殖的变化.结果 酶切和DNA测序证实Tet-on基因表达系统反应质粒PTRE-HIF-1α构建成功.获得了受强力霉素调控、稳定表达HIF-1α的肝癌细胞.随着强力霉素浓度增加,hiF-1α基因表达增加,HIF-1α在体外明显增加HepG2细胞增殖活性;G0/G1期细胞指数减少,细胞增殖指数增多.RT-PCR检测结果表明,随着HIF-1α基因表达增加,Cyclin A mRNA表达增加(P＜0.001),Cyclin D1和Cyclin E mRNA表达水平无明显变化(P＞0.05).结论 HIF-1α基因在体外可以通过HIF-1α表达水平的增加而促进肝癌细胞增殖活性,通过CyclinA表达增加缩短了肝癌细胞增殖周期.     

Insertion of a suicide gene into an immortalized human hepatocyte cell line

For developing a bioartificial liver (BAL) device, an attractive alternative to the primary human hepatocytes would be the use of highly differentiated immortalized human hepatocytes with a safeguard. To test the feasibility, the primary human hepatocytes were immortalized by a plasmid SV3neo encoding simian virus 40 large T antigen (SV40Tag) gene. A highly differentiated hepatocyte line OUMS-29 was established. A suicide gene of herpes simplex virus-thymidine kinase (HSV-TK) was retrovirally introduced into OUMS-29 cells as a safeguard for clinical application. One of the resulting HSV-TK-positive cell lines, OUMS-29/tk, grew in chemically defined serum-free medium with the gene expression of differentiated liver functions. OUMS-29/tk cells were 100 times more sensitive to ganciclovir compared with unmodified OUMS-29 cells in in vitro experiments. We have established a tightly regulated immortalized human hepatocyte cell line. Essentially unlimited availability of OUMS-29/tk cells may be clinically useful for BAL therapy.     

猿肾病毒40大T抗原基因永生化大鼠星形胶质细胞株的构建

目的 构建永生化大鼠星形胶质细胞，为转基因细胞移植镇痛提供细胞载体。方法 采用差速粘附法体外分离和培养大鼠大脑皮层星形胶质细胞。利用脂质体将含有猿肾病毒40大T抗原(SV40Tag)基因的质粒pCMVSV40T／PUR转染培养的大鼠星形胶质细胞。经嘌呤霉素1．5μg／ml筛选后，挑选阳性细胞克隆扩大培养并连续传代。PCR、RT-PCR及免疫组化检测阳性细胞克隆中SV40Tag基因的整合情况及其表达，并对传代细胞的胶质原纤维酸性蛋白(GFAP)进行检测。结果 成功获得体外培养的大鼠星形胶质细胞，GFAP阳性表达；筛选获得阳性细胞克隆，连续传代培养近50代；PCR、RT-PCR产物经1．5％琼脂糖凝胶电泳分析显示558bp处有一特异性扩增条带，与阳性对照条带相同，而未转染pCMVSV40T／PUR的细胞无扩增条带，回收片段经测序、比对与SV40Tag的基因序列一致(100％)；同时转染的阳性细胞克隆SV40Tag和GFAP免疫染色阳性。结论 成功地构建了SV40Tag基因永生化的大鼠星形胶质细胞株。