AML1-ETO 真核表达载体的构建及对 U937细胞增殖和分化的影响

目的 构建pcDNA3.1-AML1-ETO真核表达载体,并观察AML1-ETO融合蛋白在U937细胞内的表达并检测其对细胞增殖与分化的影响.方法 以原核表达载体pCMV5-AML1-ETO为模板扩增AML1-ETO目的 片段,将该基因重组于pcDNA3.1/V5-His-TOPO真核表达载体上,用脂质体转染技术将其导入 U937细胞,经G418筛选获得稳定转染的克隆,PCR检测AML1-ETO基因的整合,RT-PCR及 Western blot检测AML1-ETO mRNA和蛋白的表达,用锥虫蓝拒染人工计数法观察细胞增殖活性,流式细胞术检测髓系分化抗原表达的变化,瑞特染色法观察细胞形态改变.结果 pcDNA3.1-AML1-ETO经酶切鉴定及DNA测序证实序列完全正确,筛选出AML1-ETO基因高表达的亚克隆,证实AML1-ETO基因稳定转染到U937细胞中并得到表达;转染细胞增殖受抑(P<0.05),髓系分化抗原CD11b表达阳性率[(4.17±0.31)%]低于转染空载体和未转染对照组[(11.40±0.17)%、(11.03±0.15)%](P<0.001),形态呈低分化表现.转染细胞经TPA处理后,CD11b表达无明显变化(P>0.05).结论 成功构建pcDNA3.1-AML1-ETO表达载体,并在真核细胞中得到了正确表达,AML1-ETO 基因能抑制U937细胞的增殖与分化,为进一步研究该基因致白血病的机制奠定了基础.

Abstract：

Objective To construct a pcDNA3. 1 -AML1-ETO expression vector and investigate its effects on proliferation and differentiation of U937 leukemic cells. Methods AML1 -ETO gene was amplified by PCR from pCMV5-AML1-ETO and inserted into eukaryotic expression plasmid pcDNA3. 1/V5-His-TOPO. The recombinant plasmid was transfected into U937 cells by Lipofectamin 2000. Individual clones selected with G418 were isolated. The integration and the expression levels of AML1-ETO in transfectants were determined by PCR, RT-PCR and Western blot analysis respectively. Trypan blue refusal staining method was used to detect the proliferation of U937 cells. Light microscope was applied to observe the morphologic changes of the cell. The expression of myeloid cell differentiation antigen was detected using flow cytometry. Results The recombinant pcDNA3. 1-AML1-ETO was confirmed by enzyme digestion and sequencing. The highly expressing AML1-ETO subclone was established. AML1-ETO was expressed in U937 cells transfected with pcDNA3.1 -AML1-ETO. The growth of the monoclonal cells was inhibited evidently (P < 0. 05). The expression of CD11b in transfected group [(4. 17±0. 31)%] was lower than that in empty plasmid transfected group and non-transfected group [(11.40 ± 0. 17)% and (11.03 ± 0. 15) %] respectively (P < 0.001). Transfected cells displayed morphology of less differentiation. The expression level of CD11b was unchanged in transfected cells treated with TPA (P > 0. 05). Conclusion The eukaryotic expression vector for AML1ETO gene was successfully constructed and expressed in U937. AML1-ETO inhibits the proliferation and differentiation of transfected cells. It provides the basis for further study of mechanisms of AML1 -ETO in leukemogenesis.     

肾缺血对缺氧诱导microRNAs及VEGF-NOTCH信号分子的影响

Objective To investigate the expression changes of microRNAs and VEGF-NOTCH in renal ischemic injury in mice, and to explore the potential mechanism associated with renal angiogenesis.Method Male Balb/c mice were subjected to a standard renal ischemia to induce acute kidney injury (AKI) after 45 min of bilateral renal artery clamping. Following 4 h, 24 h of reperfusion or sham operation, kindey tissues were collected and subjected to detect the expression changes of microRNAs which relatived with angiogenesis and VEGF, Flk-1, Notch1 mRNA by Quantitative Real-time RT-PCR. Flk-1 protein was detected by Western blotting analysis at 24 h and 72 h following Ischemia/Reperfusion(I/R) injury. The expression of CD31 was examined in tissue sections by immunohistochemistry staining, and the microvessels in ischemic region of each group were counted. Results miRNA-210 and miRNA-92a expression increased significantly, with prominent changes at 4 h and 24 h after reperfusion( P ＜ 0.05 ). VEGF and Flk-1 mRNA expression and Flk-1 protein were increased in renal I/R compared with control group respectively (P＜0.05 ).Immunohistochemistry staining results of CD31 showed a significant increase of microvessels in renal ischemic region. Conclusion This study first reported the changes in miRNAs expression in response to kidney I/R in mouse. our results implied that miRNAs may be involved in targeting VEGF-Notch pathway signaling to regulate angiogenesis after renal I/R injury. It provided novel insights into the angiogenesis mechanism of renal ischemic injury.     

大鼠肾缺血/再灌注损伤不同时相Fas、FasL蛋白表达及意义

AIM: To study the relationship between the expression of Fas/FasL protein and apoptosis in rats after renal ischemia/repernision. METHODS: Establish the models of renal ischemia/reperfusion injury in rat and SABC im-munohistochemical methods were used to detect the changes of expression of Fas/FasL protein. Pathomorphological changes in renal ischemia/reperfusion injury were observed. RESULTS: The expression of Fas/FasL proteins was negative in the sham operated group. Fas/FasL proteins were increased in renal in ischemia/reperfusion group, and gradually upregulated with the duration of ischemia or reperfusion and peaked at 72 h of reperfusion. The expression of Fas/FasL proteins was atronger in 60 min ischemia group than 30 min ischemia group and they were mainly expressed in renal tubule. We observed local necrosis and inflammatory cells infiltration around infracted area in ischemia/repernision group by HE dyeing methods. And the necrosis area was mainly occurred around proximal convoluted tubule. CO     

多器官功能障碍综合征大鼠肺组织血栓调节蛋白和基质金属蛋白酶-9的表达

Objective To determine the expressions of thrombomodulin (TM) and matrix metalloproteinase-9(MMP-9) in the lung of rats with multiple organ dysfunction syndrome (MODS) and to investigate the mechanism of lung injury in MODS. Method Forty adult mule Sprague-Dawley (SD) rats were randomly divided into two groups,namely the normal control group and the MODS model group. The rats of model group were further divided into four subgroups as per different intervals (6 h, 12 h, 24 h and 48 h) ,and there were 8 rats in each groups. The animal models of MODS were estabhshed by two hits,the left eyeball of each model rat was removed to bleed to 2 mL/100g,and four hours later, lipopolysaccharide (LPS 5 mg/kg) was injected into intraperitoneal cavity of model rats. The same volume of saline was injected intraperitoneally into rats of control group instead of LPS. All rats were sacrificed at various intervals. The histological changes in lung tissue were observed by naked eye and light microscope. The expressions of TM and MMP-9 proteins were deteceted by using immunohistechemistry. One-way ANOVA was used for comparison among multiple group. Results (1)There were no histopathological changes in lung of rats of control group, and the lung injury was serious in MODS rots. (2) Compared with the rots of con-trol group, the expression of TM in lung tissue of MODS rats increased 6 hours after LPS, reached peak 12 hours later(P <0.01),and then decreased during 24～48 period.There was no significant difference in expression of TM between two groups 48 hours later. Compared with control group, the expressions of MMP-9 in lung tissue of MODS rats didn't significantly increase 6 ～ 48 hours after LPS (P < 0.01). Conclusions There are endothelium damage and extracellular matrix damage found in the lung tissue of rats at the early phase of MODS. TM and MMP-9 are good biomarkers of endothelium and extracellular matrix damage, and they can be used for diagnosing and es-timating the severity of injury lungs at the early phase of MODS.     

Changes of expression of Fas and FasL protein in rats after renal ischemia/reperfusion injury

AIM：To study the relationship between the expression of Fas/FasL protein and apoptosis in rats after renal ischemia/reperfusion.METHODS:Establish the models of renal ischemia/reperfusion injury in rat and SABC immunohistochemical methods were used to detect the changes of expression of Fas/FasL protein.Pathomorphological changes in renal ischemia/reparfusion injury were observed.RESULTS:The expression of Fas/FasL proteins was negative in the sham operated group.Fas/FasL proteins were increased in renal in ischemia/reperfusion group,and gradually upregulated with the duration of ischemia or reperfusion and peaked at 72h of reperfusion.The expression of Fas/FasL proteins was stronger in 60min ischemia group than 30min ischemia group and they were mainly expressed in renal tubule.We observed local necrosis and inflammatory cells infiltration around infracted areain ischemia/reperfusion group by HE dyeing methods.And the necrosis area was mainly occurred around proximal convoluted tubule.CONCLUSIONS:These findings suggested that Fas/FasL proteins were over expressed after ischemia/reperfusion injury in rats renal.And Fas/FasL system was involved in the process of renal ischemia/reperfusion injury.     

RNA干扰靶向抑制结缔组织生长因子拮抗肾脏纤维化的发展

Objective To investigate the impact on renal fibrosis by inhibition of connective tissue growth factor( CTGF) by RNA interference in spontaneous hypertension rat( SHR) . Method Twenty SHR were randomly (random number) divided into SHR group ( n = 10) and RNAi group ( n = 10), eight Wistar-Kyoto rats were set as control. At the end of RNA interference procedure, all the rats were sacrificed and the kidneys were harvested. The mRNA and plasmosin of CTGF and fibronectin(FN) of renal tissue were extracted and measured by RT-PCR and Western Blotting. And the localization of CTGF and FN were analyzed with immunohistochernistry technique. The collagen deposition(shown as collagen volume traction, CVF) were evaluated with 0.1% sirius-picric staining, and the hydroxyproline of myocardium were detected by colorimetry. Results The mRNA and protein expression of CTGF decreased 66% and 62% in RNAi group (P < 0.01). The mRNA and protein expression of FN decreased 56% and 51% in RNAi group.The same inhibition effect was observed by hislological analysis. Immuno-histochemistry showed that CTGF localized both in renal parenchyma and renal interstitium, whereas FN majorly expressed in renal interstitium. Observation with light microscope showed that collagen deposition(CVF)decreased sharply in RNAi group versus SHR group. And the same effect was viewed in hydroxypnoline assay[SHR group: (0.596 ± 0.067) μg/mg, RNAi group: (0.368±0.084) μg/mg, P < 0.01 ] .Further study by polarized microscope displayed that RNA interference mainly suppressed type I collagen synthesis. Conclusions Targeted inhibition of CTGF by RNA interference leads significant decrease of extracellular matrix deposition in kidney. And the anti-fibrotic effect independent of lower the blood pressure. This study indicated CTGF take a key role in the development and progress of renal fibrosis.     

地塞米松对脑损伤大鼠核转录因子-κB水平的影响

Objective To explore the effects of dexamethasone on nuclear factor-kB (NF-κB) expression in brain tissue after traumatic brain injury (TBI). Methods Forty rats were randomly divided into two groups: dexamethasone treatment and no treatment, and severe brain injury was produced by gas percussion in both groups. At 0, 6, 24, 72 and 120 hours after injury, 5 rats of each group were executed and the histopathological changes in brain tissue in rats were observed by hematoxylin-eosin (HE) stain. The expression of NF-κB in brain tissue of rats was detected by immunohistochemical method. Results NF-κB expression was significantly up-regulated at 6 hours in brain tissue of rats after TBI (P<0.05), reaching the highest level at 24 hours (P<0. 01). It showed a tendency to lower, but was still high at 120 hours after TBI (P<0. 05 or P<0. 01). After treatment with dexamethasone, NF-κB level was lowered at 6, 24 and 72 hours (all P<0. 01). Conclusion NF-κB expression is up-regulated in brain tissue in early period after TBI, and keeps on a high level, thus inducing inflammatory response to produce secondary injury to brain tissue. Dexamethasone shows protective effects by regulating the levels of NF-κB and prevents secondary injury which is caused by the inflammatory cytokines in rat brain tissue after TBI.     

RNA干扰靶向抑制结缔组织生长因子拮抗肾脏纤维化的发展

Objective To investigate the impact on renal fibrosis by inhibition of connective tissue growth factor( CTGF) by RNA interference in spontaneous hypertension rat( SHR) . Method Twenty SHR were randomly (random number) divided into SHR group ( n = 10) and RNAi group ( n = 10), eight Wistar-Kyoto rats were set as control. At the end of RNA interference procedure, all the rats were sacrificed and the kidneys were harvested. The mRNA and plasmosin of CTGF and fibronectin(FN) of renal tissue were extracted and measured by RT-PCR and Western Blotting. And the localization of CTGF and FN were analyzed with immunohistochernistry technique. The collagen deposition(shown as collagen volume traction, CVF) were evaluated with 0.1% sirius-picric staining, and the hydroxyproline of myocardium were detected by colorimetry. Results The mRNA and protein expression of CTGF decreased 66% and 62% in RNAi group (P < 0.01). The mRNA and protein expression of FN decreased 56% and 51% in RNAi group.The same inhibition effect was observed by hislological analysis. Immuno-histochemistry showed that CTGF localized both in renal parenchyma and renal interstitium, whereas FN majorly expressed in renal interstitium. Observation with light microscope showed that collagen deposition(CVF)decreased sharply in RNAi group versus SHR group. And the same effect was viewed in hydroxypnoline assay[SHR group: (0.596 ± 0.067) μg/mg, RNAi group: (0.368±0.084) μg/mg, P < 0.01 ] .Further study by polarized microscope displayed that RNA interference mainly suppressed type I collagen synthesis. Conclusions Targeted inhibition of CTGF by RNA interference leads significant decrease of extracellular matrix deposition in kidney. And the anti-fibrotic effect independent of lower the blood pressure. This study indicated CTGF take a key role in the development and progress of renal fibrosis.     

地塞米松对脑损伤大鼠核转录因子-κB水平的影响

Objective To explore the effects of dexamethasone on nuclear factor-kB (NF-κB) expression in brain tissue after traumatic brain injury (TBI). Methods Forty rats were randomly divided into two groups: dexamethasone treatment and no treatment, and severe brain injury was produced by gas percussion in both groups. At 0, 6, 24, 72 and 120 hours after injury, 5 rats of each group were executed and the histopathological changes in brain tissue in rats were observed by hematoxylin-eosin (HE) stain. The expression of NF-κB in brain tissue of rats was detected by immunohistochemical method. Results NF-κB expression was significantly up-regulated at 6 hours in brain tissue of rats after TBI (P<0.05), reaching the highest level at 24 hours (P<0. 01). It showed a tendency to lower, but was still high at 120 hours after TBI (P<0. 05 or P<0. 01). After treatment with dexamethasone, NF-κB level was lowered at 6, 24 and 72 hours (all P<0. 01). Conclusion NF-κB expression is up-regulated in brain tissue in early period after TBI, and keeps on a high level, thus inducing inflammatory response to produce secondary injury to brain tissue. Dexamethasone shows protective effects by regulating the levels of NF-κB and prevents secondary injury which is caused by the inflammatory cytokines in rat brain tissue after TBI.     

地塞米松对脑损伤大鼠核转录因子-κB水平的影响

Objective To explore the effects of dexamethasone on nuclear factor-kB (NF-κB) expression in brain tissue after traumatic brain injury (TBI). Methods Forty rats were randomly divided into two groups: dexamethasone treatment and no treatment, and severe brain injury was produced by gas percussion in both groups. At 0, 6, 24, 72 and 120 hours after injury, 5 rats of each group were executed and the histopathological changes in brain tissue in rats were observed by hematoxylin-eosin (HE) stain. The expression of NF-κB in brain tissue of rats was detected by immunohistochemical method. Results NF-κB expression was significantly up-regulated at 6 hours in brain tissue of rats after TBI (P<0.05), reaching the highest level at 24 hours (P<0. 01). It showed a tendency to lower, but was still high at 120 hours after TBI (P<0. 05 or P<0. 01). After treatment with dexamethasone, NF-κB level was lowered at 6, 24 and 72 hours (all P<0. 01). Conclusion NF-κB expression is up-regulated in brain tissue in early period after TBI, and keeps on a high level, thus inducing inflammatory response to produce secondary injury to brain tissue. Dexamethasone shows protective effects by regulating the levels of NF-κB and prevents secondary injury which is caused by the inflammatory cytokines in rat brain tissue after TBI.     

大鼠Delta1基因真核表达载体的构建与鉴定

背景:最近有数据表明,Notch信号通路在外周移植免疫应答反应中发挥重要的调节作用,可以促进调节性T的分化,诱导抗原特异性的免疫耐受.推测Notch/Notch配体可能在MHC:TCR界面发挥作用.目的:构建大鼠Deltal基因(Notch配体)真核表达载体,并观察其在树突状细胞中的表达.方法:应用RT-PCR方法从大鼠骨髓细胞中获得全长Delta1基因片段,并将此基因片段构建在pcDNA3.1(+)真核表达载体上.利用脂质体基因转染技术将含大鼠Delta1基因片段的真核表达载体pcDNA3.1(+)/Delta1转染入树突状细胞中,观察Delta1基因在树突状细胞中的表达.结果与结论:双酶切鉴定结果显示,Delta1已成功构建在pcDNA3.1的Hindlll和Xbal双酶切位点之间,任选一个酶切阳性克隆pcDNA3.1/Deltal送上海生工生物公司进行序列测定,测序结果与Genebank中Delta1基因序列完全一致,阅读框正确.转染Delta1基因的树突状细胞形态与其亲本细胞类似,蛋白免疫印迹法检测到细胞内Deltal的表达显著增高.实验成功利用基因转染方法将Delta1基因导入树突状细胞,构建了真核表达载体pcDNA3.1(+)/Delta1,并使之高效表达并分泌Delta1蛋白.     

人血管内皮生长因子165基因转染兔骨髓间充质干细胞的蛋白表达

背景:骨髓间充质干细胞是最好的组织工程种子细胞来源,含有血管内皮生长因子165(vascular endothelial growth factor 165,VEGF165)不仅对血管再生和启动成骨修复有重要意义,其持续稳定的释放还能够提高新生骨的矿化程度,增强修复组织的力学性能。目的:观察hVEG F165基因转染的兔骨髓间充质干细胞分泌血管内皮生长因子的蛋白功能。方法:体外分离、培养兔骨髓间充质干细胞,纯化并鉴定兔骨髓间充质干细胞;免疫荧光法检测细胞表面标志;传代培养后的骨髓间充质干细胞以pcDNA3.1-VEGF165质粒和脂质体1:3比例的混合液转染,并分为3组:转染组应用pcDNA3.1-VEGF165转染细胞,空载体转染组应用pcDNA3.1-空载体转染,未转染组不处理。通过ELISA和Western-blot检测转染后细胞中外源性血管内皮生长因子的表达。结果与结论:转染组与其他两组比较,VEGF165蛋白含量显著增高,差异有显著性意义(P<0.05),但空载体转染组与未转染组之间差异无显著性意义(P>0.05),转染组不同时间点之间VEGF165蛋白含量差异均有显著性意义(P<0.05),hVEGF165基因转染的骨髓间充质干细胞能成功分泌VEGF165蛋白。提示采用基因转染技术可将hVEGF165基因转染到骨髓间充质干细胞中并可有效表达具有生物活性的VEGF165。     

人血管内皮生长因子165基因转染兔骨髓间充质干细胞的蛋白表达

背景：骨髓间充质干细胞是最好的组织工程种子细胞来源,含有血管内皮生长因子165（vascular endothelial growth factor 165,VEGF165）不仅对血管再生和启动成骨修复有重要意义,其持续稳定的释放还能够提高新生骨的矿化程度,增强修复组织的力学性能。目的：观察hVEG F165基因转染的兔骨髓间充质干细胞分泌血管内皮生长因子的蛋白功能。方法：体外分离、培养兔骨髓间充质干细胞,纯化并鉴定兔骨髓间充质干细胞;免疫荧光法检测细胞表面标志;传代培养后的骨髓间充质干细胞以pcDNA3.1-VEGF165质粒和脂质体1：3比例的混合液转染,并分为3组：转染组应用pcDNA3.1-VEGF165转染细胞,空载体转染组应用pcDNA3.1-空载体转染,未转染组不处理。通过ELISA和Western-blot检测转染后细胞中外源性血管内皮生长因子的表达。结果与结论：转染组与其他两组比较,VEGF165蛋白含量显著增高,差异有显著性意义（P〈0.05）,但空载体转染组与未转染组之间差异无显著性意义（P〉0.05）,转染组不同时间点之间VEGF165蛋白含量差异均有显著性意义（P〈0.05）,hVEGF165基因转染的骨髓间充质干细胞能成功分泌VEGF165蛋白。提示采用基因转染技术可将hVEGF165基因转染到骨髓间充质干细胞中并可有效表达具有生物活性的VEGF165。     

大鼠组织因子基因克隆及其在C6细胞系中的表达

目的:克隆大鼠组织因子(tissue factor,TF)基因,构建真核表达载体pEGFP-N1-TF,转染C6大鼠神经胶质瘤细胞并检测转染细胞内TF表达水平.方法:采用RT-PCR法从Wistar大鼠肺组织中扩增TF基因,克隆到真核表达载体pEGFP-N1上.通过测序鉴定重组质粒中插入TF的完整性和可靠性.应用脂质体法将鉴定正确的重组质粒转入C6细胞中,荧光显微镜下观察EGFP报告基因的表达强度和转染效率,并对转染细胞的TF-eGFP融合蛋白进行Western blot检测.结果:成功构建pEGFP-N1-TF真核表达载体,转染C6细胞24 h后,在荧光显微镜下可以观测到荧光,并通过Western blot技术检测到TF-eGFP融合蛋白的表达.结论:重组真核表达载体pEGFP-N1-TF构建成功,转染C6细胞后获得了良好的瞬时表达.     

人核心蛋白聚糖基因靶向肺癌细胞特异性表达载体的构建及其表达

目的构建调控人核心蛋白聚糖基因特异性在肺癌细胞A549内表达的基因表达载体,为进一步应用该基因进行肺癌靶向治疗的研究奠定实验基础。方法应用PCR技术从人外周血基因组中扩增肺癌细胞特异性表达的人分泌型白细胞蛋白酶抑制剂的启动子,利用基因重组技术将该启动子插入真核细胞表达载体pcDNA3.1（＋）,替换该载体的CMV启动子与增强子序列,从而构建成由人分泌型白细胞蛋白酶抑制剂基因的启动子启动基因表达的基因表达载体。将人核心蛋白聚糖基因通过基因重组技术插入到上述载体人分泌型白细胞蛋白酶抑制剂基因启动子的下游。PCR产物通过测序鉴定核苷酸序列。重组载体通过限制性酶切进行鉴定。结果 PCR扩增的人分泌型白细胞蛋白酶抑制剂的启动子片段长度为1 250 bp;该启动子经测序获得的核苷酸序列与Genebank上该基因上游5′末端转录调控区的序列完全一致;重组载体的酶切鉴定结果显示（1）该启动子成功插入到pcDNA3.1（＋）载体,并替换CMV启动子与增强子序列,（2）人核心蛋白聚糖基因成功插入该载体。结论成功构建由人分泌型白细胞蛋白酶抑制剂基因启动子调控人核心蛋白聚糖基因表达载体,该载体实现调控人核心蛋白聚糖基因在肺癌细胞内特异性表达。     

重组pcDNA3.1-hBMP-2转染兔脂肪干细胞体外诱导的成骨分化

背景：骨形态发生蛋白2具有很强的诱导干细胞成骨活性。目的：构建人骨形态发生蛋白2基因真核表达载体,探讨其转染脂肪干细胞后的成骨效果。方法：通过噬菌斑原位杂交筛选人混合细胞cDNA文库获得人骨形态发生蛋白2基因,与真核表达载体pcDNA3.1-连接,构建重组质粒pcDNA3.1-hBMP-2。利用脂质体LipofectamineTM2000分别介导人骨形态发生蛋白2基因、EGFP基因转染第4代脂肪干细胞,并经G418进行筛选。结果与结论：酶切鉴定及DNA测序结果证实重组质粒pcDNA3.1-hBMP-2构建成功。经计算脂质体介导的脂肪干细胞瞬时转染率为（18.0±0.42）%,并经过G418筛选后获得了稳定转染的细胞。细胞生长曲线表明转染后对脂肪干细胞生长、增殖无明显影响。ELISA检测发现人骨形态发生蛋白2组的人骨形态发生蛋白2因子表达量均高于EGFP组及未转染组,且能够稳定表达。经人骨形态发生蛋白2基因转染的脂肪干细胞的Ⅰ型胶原含量、碱性磷酸酶活性以及钙结节数目均比EGFP组及未转染组有明显升高。     

pcDNA 3.1-AKR1B10真核表达载体的构建

从胰腺癌PaTu8988S细胞中提取总RNA,并克隆AKR1B10基因,构建pcDNA 3.1-AKR1B10真核表达载体。转化感受态大肠杆菌DH5α细胞,提质粒,酶切及测序鉴定,pcDNA 3.1-AKR1B10真核表达载体构建成功。     

构建人HCN4基因重组腺病毒载体及其对大鼠骨髓间充质干细胞的感染效率

背景:超极化激活及环化核苷酸门控阳离子通道(hyperpolarization-activated cyclic nucleotide-gated cation channel,HCN)基因由于具有不增加诱发心律失常的风险、能够接受自主神经系统调节等优势,成为目前最受关注的生物起搏备选基因.目的:构建携带人HCN4基因的重组腺病毒载体,并测定其对大鼠骨髓间充质干细胞的感染效率.设计、时间及地点:细胞-基因学体外实验,于2008-02/09在中山大学附属第二医院林百欣实验中心完成.材料:SD大鼠10只,由中山大学实验动物中心提供.携带目的基因人HCN4 cDNA的质粒pcDNA3.1-HCN4、人胚肾293细胞、大肠杆菌DH5α由中山大学附属第二医院林百欣实验中心保存.腺病毒穿梭质粒pShuttle-CMV、骨架质粒pAdxsi购自北京诺赛基因组研究中心有限公司.方法:质粒pcDNA3.1-HCN4用Hind Ⅲ+Xba Ⅰ双酶切后回收HCN4片段,亚克隆至pShuttle-CMV中,得到重组穿梭质粒;1-Ceu Ⅰ+1-Sce Ⅰ双酶切处理pShuttle-CMV-HCN4,回收CMV-HCN4片段,亚克隆至腺病毒骨架载体pAdxsi,得到重组腺病毒质粒;重组腺病毒质粒酶切线性化后,应用脂质体法转染293细胞进行包装扩增,得到重组腺病毒AdHCN4;应用AdHCN4转染大鼠骨髓间充质干细胞.主要观察指标:重组腺病毒质粒载体的鉴定,重组腺病毒的鉴定及滴度测定,重组腺病毒的感染效率.结果:构建的重组穿梭质粒pShuttle-CMV-HCN4用Hind Ⅲ+Xho Ⅰ双酶切,得到大小为3 600 bp(HCN4)和5 100 bp(pshutIle-CMV)两个片段,DNA测序结果证实人HCN4基因的全长序列已正确插入到pShuttle-CMV穿梭质粒中;重组腺病毒质粒pAdxsi-CMV-HCN4用Xho Ⅰ酶切得到7个片段,而作为对照的空腺病毒质粒只得到6个片段;重组腺病毒质粒在293细胞中包装后产生的重组腺病毒对293细胞有致病作用;重组腺病毒AdHCN4 PCR鉴定可见657 bp的阳性扩增条带;经多次重复感染后,病毒滴度检测达2.5×10~(11) PFU/mL.成功转染AdHCN4的大鼠骨髓间充质干细胞可表达绿色荧光蛋白,当病毒感染复数值为800,转染效率最高,达90%.结论:实验成功构建携带人HCN4基因的重组腺病毒载体,并可在体外有效转染大鼠骨髓间充质干细胞.     

外源Smad7转染肝星状细胞及对转化生长因子β1和Ⅰ、Ⅲ型胶原mRNA表达的影响

背景:Smad7是转化生长因子β信号转导途径的主要抑制性蛋白,具有抗纤维化的作用.目的:构建并鉴定大鼠Srnad7真核表达质粒,观察外源Smad7可否有效转染肝星状细胞T6,并进一步研究其对转化生长因子β及Ⅰ、Ⅲ型胶原mRNA表达水平的影响.设计、地点:基因重组及细胞观察实验,于新疆石河子大学医学院第一附属医院完成.材料:pcDNA3.1(+)质粒为课题组保留;大肠杆菌DH5a系石河子大学医学院新疆地方与民族高发病教育部重点实验室所赠;肝星状细胞T6细胞由中国医学科学院肿瘤医院肿瘤研究所提供品.方法:采用基因重组技术将Smad7cDNA插入真核表达载体pcDNA3.1(+),构建大鼠Smad7真核表达质粒.脂质体介导转染肝星状细胞T6细胞,分为正常对照、空质粒及转染组,G418筛选,挑取阳性细胞.主要观察指标:反转录-聚合酶链反应法检测各组中Smad7、转化生长因子β及Ⅰ、Ⅲ型胶原mRNA的表达水平.结果:酶切和测序结果证实Smad7真核表达质粒构建成功.Smad7转染组与正常对照组、空质粒组比较:Smad7 mRNA 达显著增加(P<0.01);转化生长因子β、Ⅰ型胶原mRNA表达减少(P<0.01);Ⅲ型胶原mRNA表达差异无显著性意义(P>0.05).正常对照组、空质粒组smad7、转化生长因子β及Ⅰ、Ⅲ型胶原mRNA表达差异无显著性意义(P值均>0.05).结论:大鼠Smad7真核表达质粒构建成功,外源Srnad7转染肝星状细胞T6细胞后可有效表达,并能降低转化生长因子β及Ⅰ型胶原mRNA表达水平.     

SOX-9和胰岛素样生长因子1共同转染大鼠骨髓间充质干细胞向软骨细胞的分化

背景:单基因诱导虽能对骨髓间充质干细胞向软骨细胞分化产生积极影响,但作用有限,多种基因共同作用才符合机体真实内环境的需求,并有助于发挥多基因产物之间的协同作用,提高分化效果.目的:验证应用SOX-9和胰岛素样生长因子1基因转染大鼠骨髓间充质干细胞后,目的基因分泌情况及向软骨细胞的分化效果.设计、时间及地点:两样本观察,实验于2008-04/2009-02在中国医科大学细胞生物实验室完成.对象:6周龄健康雄性Wistar大鼠2只用于骨髓间充质干细胞提取.方法:扩增、提取质粒pcDNA3.1-IGF-1、pcDNA3.1-SOX-9.分离、纯化Wistar大鼠骨髓间充质干细胞.按转染情况分为5组:①未转染组:仅加入双无(无血清无双抗)L-DMEM.②转染窄载体组:双无L-DMEM+pcDNA3.1空载体和脂质体.③转染SOX-9组:双无L-DMEM+pcDNA3.1-SOX-9质粒和脂质体.④转染胰岛素样生长因子1组:双无L-DMEM分别+pcDNA3.1-IGF-1质粒和脂质体.⑤共转染组:双无L-DMEM分别+pcDNA3.1-IGF-1、pcDNA3.1-SOX-9质粒和脂质体,混合.主要观察指标:对转染后细胞进行筛选,MTT法测定筛选后细胞的增殖活性,反转录-聚合酶链反应和Western blot法检测筛选后细胞目的基因和蛋白的表达.结果:MTT法检测转染SOX-9组、转染胰岛素样生长因子1组和共转染组骨髓间充质干细胞增殖活性A值显著高于未转染组、转染空载体组(P<0.01).反转录-聚合酶链反应和Westem blot检测目结果显示,SOX-9表达以转染SOX-9组最多;胰岛素样生长因子1表达以共转染组最多:软骨细胞特异性基质Ⅱ型胶原表达以共转染组最多.结论:双基因转染在诱导骨髓间充质干细胞向软骨细胞转化的能力和细胞外基质分泌的能力上更明显高于单基质转染;另外结果间接说明胰岛素样生长因子1具有诱导骨髓间充质干细胞向软骨细胞分化的作用,但能力弱于SOX-9.