慢病毒载体介导hTERT基因修饰的人羊膜上皮细胞重建角膜表层的实验研究
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摘要
组织工程化角膜上皮需要合适的种子细胞,人羊膜上皮细胞(human amnioticepithelium cells,hAECs)容易获得、取材方便,在伦理问题上也不存在任何争议,经研究发现hAECs具有类似胚胎干细胞或多能干细胞的多向分化潜能,在不同生长因子和微环境调控下能分化成多种不同组织的细胞,其极可能诱导分化成角膜上皮细胞。因此,hAECs可能是组织工程角膜上皮一种新的细胞来源。但是由于hAECs属于终末分化细胞,体外生存期短、培养困难,限制了其广泛应用。有鉴于此,本研究利用慢病毒载体携带目的基因人端粒酶催化亚基(hTERT)和标记基因增强型绿色荧光蛋白(EGFP)组成的融合基因转染hAECs,使其生长周期延长甚至达到永生化,然后应用转基因hAECs构建复层上皮细胞-角膜基质移植材料重建角膜表层,探讨转基因hAECs做为组织工程化角膜表层一种新的细胞来源的应用价值。
第一部分人羊膜上皮细胞(hAECs)的体外培养和其生物学鉴定
目的:
研究人羊膜上皮细胞(hAECs)体外培养的生物学特性,探讨hAECs体外培养的方法及其干细胞特性,为基因转染hAECs并进行角膜表层重建奠定实验基础。
方法:
1采用酶消化法体外分离培养hAECs,观察细胞在体外培养条件下的生长情况,并对生长良好的原代细胞进行消化传代,在光镜下对细胞进行形态学观察和HE染色观察;
2对传代的hAECs进行细胞角蛋白CK7、CK8和CK18免疫组化染色鉴定及免疫荧光检测hAECs中干细胞转录因子OCT-4的表达;
3对传代的hAECs进行流式细胞仪检测干细胞表面分子标记CD29、CD34、CD44、CD45、CD105的表达。
4免疫组化检测传代hAECs中端粒酶催化亚基hTERT蛋白的表达。
结果:
1在体外培养条件下,hAECs能贴壁生长,细胞呈典型的上皮细胞样外观,正常情况下可传7—8代。HE染色可见细胞大小较均一,呈不规则的多角形,细胞连接成片。
2免疫组化结果显示培养的hAECs胞浆中见CK7、CK8、CK18单克隆抗体染色阳性,OCT-4免疫荧光结果显示hAECs的胞浆中有荧光表达。
3流式细胞仪检测发现hAECs干细胞标记分子CD29、CD34的表达为阳性,但干细胞标记分子CD44、CD45、CD105的表达为阴性。
4免疫组化结果显示培养的hAECs胞浆中hTERT单克隆抗体染色阴性。
结论:
1通过酶分离法可成功分离出纯度较高的hAECs,获得的细胞能在体外进行短期内培养。
2培养的hAECs是单层上皮细胞来源,并具有干细胞的某些特征。
第二部分真核表达载体pLenti6/V5-DEST-hTERT-EGFP的构建
目的:
利用慢病毒载体构建携带有目的基因人端粒酶催化亚基(hTERT)和标记基因增强型绿色荧光蛋白(EGFP)的真核表达载体pLenti6/V5-DEST-hTERT-EGFP,为基因转染hAECs奠定实验基础。
方法:
1基因重组穿梭质粒pDONR221-hTERT-EGFP的构建和鉴定
以PCI-neo-hTERT质粒为模板PCR法获取目的基因hTERT,通过BP反应,将hTERT定向克隆至pDONR221得pDONR221-hTERT;以质粒pEGFP-N1为模板,通过PCR反应得到-linker-EGFP-产物。通过双酶切,T4-DNA连接酶反应,得到pDONR221-hTERT-EGFP产物。然后转化大肠杆菌DH5α,卡那霉素筛选,挑克隆行PCR、酶切及测序鉴定。
2 pLenti6/V5-DEST-hTERT-EGFP重组载体的构建和鉴定
取pDONR221-hTERT-EGFP重组质粒和pLenti6/V5-DEST载体,进行LR重组反应,得到pLenti6/V5-DEST-hTERT-EGFP重组质粒,之后转化入大肠杆菌STB13感受态,涂含氨苄霉素的LB平板,挑取单菌落扩增培养,提取质粒,并进行测序鉴定。
3含hTERT-EGFP病毒颗粒上清的包装和收集
利用脂质体转染法将pLenti6/V5-DEST-hTERT-EGFP质粒转入293FT细胞,培养24h后,更换完全培养基,再48 h-72 h后,采用过滤法收集含病毒颗粒的上清液,一80℃保存备用。
结果:
1慢病毒入门载体pDONR221-hTERT-EGFP的构建和鉴定结果
本实验成功扩增出长达约3.4Kbp的attB1-hTERT-attB2片段,行PCR、酶切及测序鉴定,证实hTERT已克隆入入门载体pDONR221,并成功获得pDONR221-hTERT-EGFP产物。
2慢病毒入门载体pDONR221-hTERT的1547位点定点突变(将1547位点突变的碱基G变回原序列A)
测序结果表明慢病毒入门载体的目的基因hTERT存在1547位点发生错义突变,经设计含1547位点正确碱基的引物,通过定点突变技术将慢病毒入门载体pDONR221-hTERT的1547位点定点突变,经测序证实突变成功。
3慢病毒表达载体pLenti6/V5-DEST-hTERT-EGFP的构建和鉴定结果
经LR重组反应构建得慢病毒重组质粒pLenti6/V5-DEST-hTERT-EGFP,将重组质粒送去测序,证实重组质粒pLenti6/V5-DEST-hTERT-EGFP构建成功。
4含hTERT-GFP病毒颗粒上清液的制备结果
将pLenti6/V5-DEST-hTERT-GFP与Virapower~(TM)Packaging Mix利用脂质体共转染包装细胞293FT细胞,终止转染72h后收集上清液,过滤,即得含hTERT-GFP病毒颗粒的上清液。
结论:
1首次成功构建慢病毒重组质粒pLenti6/V5-DEST-hTERT-EGFP。
第三部分慢病毒表达载体pLenti6/V5-DEST-hTERT-EGFP的转染和表达
一、pLenti6/V5-DEST-hTERT-EGFP基因转染hAECs
目的:
在本实验中,利用慢病毒将目的基因人端粒酶逆转录酶基因(hTERT)和标记基因EGFP转染入人羊膜上皮细胞(hAECs),并扩增培养含hTERT-EGFP基因的克隆细胞,观察转染细胞的生长特性,用流式细胞仪检测转染细胞EGFP阳性表达率和检测hTERT基因转染对hAECs生长周期的影响,为下一步转染细胞的活体移植奠定实验基础。
方法:
1人羊膜上皮细胞的传代培养
2慢病毒载体的转染
2.1杀稻瘟菌素筛选浓度的确定
2.2慢病毒载体转染人羊膜上皮细胞
制成两孔转染实验组(转基因组)、两孔转空载对照组、两孔正常对照组,共六孔,24h后终止转染。
3筛选稳定表达的抗性细胞克隆并扩增培养
细胞终止转染后,继续培养24 h,加入含杀稻瘟菌素终浓度为1ug/ml的完全培养基,对细胞进行加压筛选,筛选10天左右,直至单个细胞克隆形成。重组细胞克隆形成后,换为不加筛选药物的DMEM完全培养基继续培养,至细胞接近融合时,消化传代,扩增培养。扩增培养时,仍加杀稻瘟菌素进行筛选。
4荧光显微镜下观察转染基因的瞬时表达
细胞终止转染后,用不加筛选药物的含10%胎牛血清的DMEM完全培养基继续培养12h、24h、48h、72h、96h,荧光显微镜下观察每个时间段瞬时转染细胞和稳定转染细胞绿色荧光蛋白的表达。
5倒置显微镜下观察转染细胞的生长形态
6流式细胞仪检测转染细胞EGFP阳性表达率
分别收集未转染的hAECs、瞬时转染12h、24h、48h、96h的hAECs,制成单细胞悬液,行流式细胞仪检测,利用cell-quesL软件分析EGFP阳性细胞表达率。
7流式细胞仪检测hTERT基因转染对hAECs生长周期的影响
分别收集稳定转染后第2代培养48h的hAECs(转基因组),常规传1代培养48h的hAECs(正常对照组),以及转染空载体传1代培养48h的hAECs(转染空载体组),调细胞浓度为1×10~6个/ml,用流式细胞仪检测标记细胞,CellQuest软件处理资料,全部数据采用SPSS11.0统计软件包进行处理,PI值比较采用单因素方差分析,以P＜0.05定为差异具有统计学意义。
结果:
1杀稻瘟菌素的抗性筛选
接种于6孔板中的人羊膜上皮细胞,当加入杀稻瘟菌素浓度为1ug/ml时,培养14d细胞全部死亡,从培养时间上考虑选1ug/ml作为杀稻瘟菌素的筛选浓度。
2倒置显微镜下所见
培养的人羊膜上皮细胞加入转染液12h后,有少数细胞从培养板上脱落下来漂浮于培养液中,大多数转染细胞筛选出的抗性克隆以及培养的克隆细胞均与正常细胞在形态上无显著差异。
3荧光显微镜下观察GFP的阳性表达
3.1瞬时转染12h后即可见发绿色荧光的人羊膜上皮细胞,但数量较少,强度教弱,每个低倍视野下可见0-2个;24h后发绿色荧光的人羊膜上皮细胞逐渐增多强度增强,每个低倍视野下可见5-8个左右;48h后发绿色荧光的人羊膜上皮细胞继续增多,每个低倍视野下可见10个以上;72h后发绿色荧光的人羊膜上皮细胞与48h的人羊膜上皮细胞差别不大;96h后发绿色荧光的人羊膜上皮细胞逐渐减少,每个低倍视野下可见5个左右,经1ug/ml的杀稻瘟菌素筛选10d后,观察有单细胞克隆出现。
3.2消化收集经1ug/ml的杀稻瘟菌素筛选获得稳定表达的人羊膜上皮细胞,接种于6孔板中扩增培养,贴壁后可见密集荧光。
4人羊膜上皮细胞pLenti6/V5-DEST-HTERT-EGFP基因转染效率检测结果流式细胞仪检测结果显示:未转染组人羊膜上皮细胞中,GFP阳性细胞数为零,瞬时转染12h、24h、48h、72h、96h组的人羊膜上皮细胞均可见GFP阳性表达,其中48h转染组的人羊膜上皮细胞GFP阳性表达率最高,为18.53%,与12h、24h、96h转染组比较差异具有统计学意义(P＜0.05),与72h转染组比较差异不显著,无统计学意义(P＞0.05)。
5细胞周期检测结果
转基因组人羊膜上皮细胞与正常对照组比较,处于S期和G2/M组的细胞比例增加,PI值升高,两者有显著性差异(P＜0.05),转空载体组人羊膜上皮细胞与正常对照组比较,PI值无显著变化(P＞0.05)。
结论:
1首次证明目的基因人端粒酶逆转录酶基因(hTERT)和标记基因EGFP经慢病毒介导转染入人羊膜上皮细胞(hAECs)后,并可在细胞内获得瞬时表达和稳定表达,且瞬时转染48h,人羊膜上皮细胞(hAECs)EGFP阳性表达率最高,为18.53%。
2转染入目的基因人端粒酶逆转录酶基因(hTERT)后,人羊膜上皮细胞(hAECs)的增值指数PI值明显升高,生长周期明显延长。
3慢病毒介导的转染方法对人羊膜上皮细胞(hAECs)的形态、分化和增殖能力等生物学特性无明显影响,再次证明是一种相对安全的基因转染方法。
二、pLenti6/V5-DEST-hTERT-EGFP基因在转基因hAECs中的表达
目的:
在本实验中,利用荧光定量PCR和Western blot方法从mRNA和蛋白质水平检测hTERT基因在转基因hAECs中的表达,并通过免疫组织化学法检测转基因hAECs中端粒酶催化亚基hTERT蛋白的表达,再通过裸鼠试验检测转染细胞的致瘤性,研究转染有hTERT基因的hAECs的生物安全性,为基因转染人羊膜上皮细胞并进行角膜表层重建奠定实验基础。
方法:
1应用荧光定量PCR(Real Time-PCR)方法检测hTERT mRNA在转染细胞中的表达
1.1总RNA提取(整个过程要求在超净台内进行)
分别提取常规传1代培养48h的hAECs(第1组,即正常对照组)、传1代培养48h的HepG2肝癌细胞(第2组,即阳性对照组)、以及稳定转染pLenti6/V5-DEST-hTERT-EGFP基因后第2代培养48h的hAECs(第3组,即稳定转染实验组)、瞬时转染48h的hAECs(第4组,即瞬时转染实验组)、用慢病毒空载体转染48h的hAECs(第5组,即空载转染对照组)的总RNA。
1.2总RNA纯度和完整性检测
1.2.1纯度检测:取双蒸水99μl加入比色杯中,再加入1μl总RNA样品,在核酸蛋白检测仪上测定OD值。
1.2.2总RNA完整性检测:取RNA样品2μl,1%琼脂糖凝胶电泳80V×30min,用凝胶成像系统观察总RNA的5s rRNA、18s rRNA和28s rRNA条带。
1.3逆转录:得1~(st) strand cDNA:
取上述5组细胞RNA液各2ul,转移到无菌PCR管中,行PCR反应。
1.4荧光定量qRT-PCR
1.4.1检测序列片段大小
1.4.2设计引物
1.4.3反应体系:2×2×SYBR green premix10μl+Primers mix0.8μl+cDNA1μl+H_2O 8.2μl,总体积为20μl;
1.4.4反应条件:95℃10min,95℃15s,65℃30s,72℃30s读板,共40个循环;
1.4.5融解曲线分析:温度55℃-95℃,每分钟读1次,每个样设置3个复孔。
2 Western blotting法检测转基因hAECs中端粒酶蛋白的表达
2.1样品的制备
取上述5组细胞,加入单去污剂裂解缓冲液,收集细胞裂解物,加入等体积凝胶加样缓冲液,离心后,将上清夜移至另一管中,待用。
2.2 SDS-聚丙烯酰胺凝胶电泳(SDS-PAGE)
2.2.1正确安装电泳槽玻璃板,加入分离胶,待分离胶完全聚合后,再在分离胶上灌注浓缩胶,快速插入干净的梳子,浓缩胶聚合后,小心移出梳子。
2.2.2将凝胶固定于电泳装置上,加入电泳缓冲液。按预定顺序加样,连接电泳装置与电流。当染料接近分离胶底部时,关闭电源。卸下玻璃板,分离玻璃板,标记凝胶方位。
2.3 Western Blotting法检测
电泳完毕后,将凝胶取下,贴于PVDF膜上,将胶、膜、滤纸、海绵和转移夹板夹成“三明治”状,进行转膜,闭膜,加一抗溶液孵育过夜。再加到二抗溶液中,摇床孵育1h。然后显色,感光,显影和定影,与之前预染的Marker对比,观察结果,拍照分析。
3免疫组化法检测转基因hAECs中端粒酶催化亚基hTERT蛋白的表达
采用链酶亲和素-过氧化物酶(SABC)法,检测盖玻片上常规培养的上述五种细胞中端粒酶催化亚基hTERT蛋白的表达情况。
4裸鼠致瘤实验
取稳定转染有目的基因hTERT的hAECs及HepG2肝癌细胞,取BALB/c—nu/nu品系裸鼠12只,6只接种转基因hAECs,6只接种HepG2肝癌细胞,于动物背部皮下无菌接种细胞悬液,屏障环境下饲养两周,观察细胞注射部位有无肿瘤生长以及肿瘤的大小。
结果:
1实时荧光定量PCR检测结果
第1组即正常对照组和第5组即空载转染对照组hTERTmRNA的相对含量为0即不表达hTERT,第2组即阳性对照组hTERT mRNA的相对含量为450,第3组即稳定转染组目的基因hTERT mRNA的相对含量为200,第4组即瞬时转染组目的基因hTERTmRNA的相对含量约为185,说明瞬时转染和稳定转染的hAECs内均有hTERTmRNA基因表达,而没有进行基因转染的hAECs和转染空载体的hAECs内无hTERTmRNA基因表达。
2 Western Blotting检测结果
第2组即阳性对照组细胞裂解产物,在相对分子量为314KD处呈现一条特异性蛋白条带,而第1组即正常对照组、和第3组即稳定转染组和第4组即瞬时转染组和第5组即空载转染对照组则无相应条带出现,这可能因为稳定转染组细胞中hTERT蛋白表达过少或没有表达。
3免疫组化检测结果
端粒酶催化亚基hTERT抗体在第1组即正常对照组、第4组即瞬时转染组和第5组即空载转染对照组的hAECs细胞胞浆中不着色,说明这些细胞不表达hTERT蛋白,而端粒酶催化亚基hTERT抗体在第2组即阳性对照组中细胞胞浆中呈深褐色着色,说明其hTERT蛋白表达呈强阳性,,TERT抗体在第3组即稳定转染组的hAECs中呈浅褐色着色,说明其hTERT蛋白表达呈弱阳性。
4裸鼠致瘤实验结果
裸鼠在屏障环境下饲养一周后,接种HepG2肝癌细胞的裸鼠注射部位即有肿瘤长出,并随着饲养天数的延长逐渐长大,而接种转基因hAECs的裸鼠注射部位无明显变化。
结论:
1首次从mRNA水平检测到hTERT基因在体外培养的hAECs中成功转染和表达,但蛋白质水平上没有检测到转染细胞中hTERT蛋白的表达,说明hTERT基因在mRNA水平和蛋白质水平的表达并不平衡。
2转染有目的基因hTERT且稳定表达的人羊膜上皮细胞目前尚未发现有致瘤性。
第四部分应用转基因hAECs重建角膜表层的研究
目的:
研究转基因人羊膜上皮细胞(hAECs)在新鲜角膜基质片上的生存情况,并将生长有转基因hAECs的新鲜角膜基质片移植到角膜缘干细胞缺乏的兔眼表面重建角膜表层,观察转基因人羊膜上皮细胞的存活情况及眼表的稳定性,探讨转基因HAECs在活体兔眼表面的转归,为基因转染hAECs并进行角膜表层重建奠定实验基础。
方法:
1制备兔角膜缘干细胞缺损模型
取实验兔12只,麻醉后,开睑器开睑,在兔右眼角膜表面滴用正庚醇数滴去除角膜上皮,环形切除角膜缘内2mm浅板层组织,剪去第3眼睑,结膜下注射庆大霉素及地塞米松,连续观察2个月,对角膜结膜化、新生血管情况进行记录。
2转基因pLenti6/V5-DEST-HTERT-EGFP人羊膜上皮细胞克隆的体外扩增培养
3新鲜兔角膜基质片的制备
取新西兰大白兔眼球,暴露角膜,吸取少量正庚醇,滴加到角膜表面去除角膜上皮层,留下光滑的角膜基质面。然后再在角膜缘处做一小板层切口,用无菌虹膜恢复器分离浅层角膜基质层。用7.5mm环钻钻取角膜基质,将其放在无菌超净台内风干,然后放入96孔板中,备用。
4在新鲜角膜基质片上种植转基因pLenti6/V5-DEST-HTERT-EGFP人羊膜上皮细胞并复层化
将转基因hAECs消化后,接种到96孔板中的新鲜角膜基质上,小心放入培养箱中静置培养。大约3天后,角膜基质片上的细胞形成单层融合时,取出基质片,放入事先放在6孔板内的带微孔滤膜、三维立体插入式细胞培养器中。置入细胞培养箱中静置培养,大约培养10天左右,自然形成上皮细胞的复层结构。
5新鲜角膜基质片上细胞的HE染色观察
新鲜角膜基质片上的细胞培养3天后和利用气液界面培养10天后,固定、脱水、包埋、切片,进行HE染色后光学显微镜观察。
6转基因hAECs移植片的眼表移植
6.1动物分组:将上述12只右眼角膜缘干细胞缺乏模型兔随机分成2组,按构建角膜移植片是否有细胞,分为:A组(对照组,n=6):新鲜角膜基质片上不种任何细胞,B组(pLenti6/V5-DEST-hTERT-EGFP基因转染组,n=6):种子细胞为含目的基因hTERT和标记基因EGFP的慢病毒载体转染的hAECs。
6.2模型兔麻醉后,开睑器开睑,用7.0mm环钻钻取兔右眼角膜中间组织,钝性分离及板层切除中央角膜组织,将转基因人羊膜上皮细胞移植片平铺于角膜创面,然后间断缝和移植片与角膜上。结膜下注射庆大霉素及地塞米松,局部涂四环素可的松眼膏,间断缝合上下睑缘。
7术后处置
术后术眼滴氧氟沙星滴眼液于结膜囊内,肌肉注射庆大霉素,并在饲料中加环胞霉素A。每日氧氟沙星滴眼液滴眼,每日观察植片的存活状况及眼表的稳定性。
8术后观察及检测
8.1大体观察
术后第一周内,每天在裂隙灯下观察,以后每周观察2-3次,连续观察2个月。对角膜水肿、混浊、结膜化、新生血管情况进行记录。
8.2移植眼角膜组织的HE染色观察
8.3移植眼角膜组织细胞角蛋白CK8、CK18、CK12免疫组化检测
结果:
1兔角膜缘干细胞缺乏模型
制备角膜缘干细胞缺乏模型术后观察,两个月后有10只兔眼表现为角膜表面结膜化,新生血管膜形成,全角膜灰白色混浊。另2只兔眼角膜没有出现明显的结膜化,但已长出大量新生血管,并逐渐向角膜中间蔓延。
2新鲜角膜基质片上细胞在倒置显微镜下所见
转基因hAECs接种到新鲜兔角膜基质片后,3h开始贴壁,12h细胞呈团块状堆积,24h后细胞呈棒状伸展,3d左右变成多角形,部分区域已连接成片。
3新鲜角膜基质片上细胞HE染色
转基因hAECs在新鲜兔角膜基质上3-5d左右形成致密的单层结构,细胞排列紧密,然后应用插入式细胞培养器形成的气.液界面培养法培养10d左右,转基因人羊膜上皮细胞能形成类似角膜上皮的3-5层复层结构,形成的复层上皮细胞与角膜基质连接紧密,不易脱落。
4.移植术后移植片的大体外观所见
A组:术后一个月观察,6只兔眼均表现为整个角膜高度水肿,角膜浑浊程度较重,有大量新生血管,角膜炎症反应重,分泌物多。术后两个月观察,角膜结膜化,角膜血管化严重,已向角膜中央侵犯。
B组:术后一个月观察,有6只兔眼角膜透明度有不同程度提高,角膜周边有新生血管,6只兔眼缝线已大部分脱落。术后两个月观察,5只兔眼角膜均恢复透明,角膜上皮光滑,只有1只兔眼角膜透明度较低,新生血管较多。
5.角膜移植片的HE染色所见
角膜上皮重建术后2月,B组角膜组织HE染色可见角膜表面上皮出现部分类似角膜上皮的复层排列,角膜基质层可见新生血管增生。
6.移植眼角膜组织免疫组化检测结果
角膜上皮重建术后2月,角膜组织免疫组化染色可见上皮细胞角蛋白CK8、CK18和角蛋白CK12均呈阳性表达,角膜表面上皮部分细胞胞浆呈棕黄色,细胞核蓝色。
结论:
1新鲜角膜基质有利于人羊膜上皮细胞形成类似角膜上皮的复层排列,且表达角膜上皮细胞特异性抗体CK12。
2转基因pLenti6/V5-DEST-hTERT-EGFP人羊膜上皮细胞可以成为重建角膜表层的一种新的种子细胞。
Engineering cornea epithelium surface layer need suitable seed cells.Researchers have found,Human amniotic epithelial cells(hAECs),with the characteristics of both embryonic stem cells and pluripotent stem cells,have the potential to differentiate into various tissue cells in particular condition.Being obtained easily and convenience,with no any ethical arguments,have much more chances to differentiate to corneal epithelial cells,So hAECs may be a new source of seed cells of tissue engineering cornea.But being terminally differentiated cells and with short duration in vitro,the hAECs were difficult to culture.This shortcoming restricted its application in cell therapy.In view of this,us used this fusion gene of destination gene hTERT and marker gene amplification green fluorescent protein(GFP) to transfect hAECs,which compositive with lentivirus vector,to prolong cells growth cycle,even immortalized them.next,utilizing this trans-gened hAECs to re-construct laminated epithelium and cornea surface layer,to investigate application value of the trans-gened hAECs as a new seed cell souce of engineering cornea epithelium surface layer.
Part One The culture and biologic identification of human amniotic epithelial cells(hAECs) in vitro
Objective:
To study the biologic characteristics of the cultured human amniotic epithelial cells(hAECs) in vitro,and to investigate the culturing method and stem cell characteristics of hAECs;to establish experimental foundation for transfecting hAECs with hTERTgene and reconstructing corneal surface layer.
Methods:
1.We used enzyme digestion to isolate and culture hAECs and observed the adherence and growth of the cells,the morphologic changes of cultured AECs were observed by HE staining,inverted microscope and light microscope.
2.The expression of cytokeratin CK7、CK8 and CK18 in AECs were detected by using immunohistochemical staining.The immunofluorescence was used to detect the expression of OCT-4 of stem cell factor in passaged hAECs.
3.Stem cell surface molecule marker CD29、CD34、CD44、CD45、CD 105 were detected by flow cytometry in passaged hAECs.
4.Using immunohistochemistry to study the expression of hTERT protein in passaged hAECs
Results:
1 hAECs could adhere and grow very well in vitro,the cells looked like slabstone, they could passage 7—8 generations in normal circumstances.
2 The results of immunohistochemistry showed The hAECs expressed cytokeratin CK7、CK8、CK18 in cytoplasm,the result of immunofluorescence showed The expression of OCT-4 of stem cell factor in hAECs was positive too.
3 Detection of stem cell surface molecule marker by the flow cytometry showed The expression of CD29、CD34 on hAECs were positive,The expression of CD44、CD45、CD 105 on hAECs were negative.
4 The results of immunohistochemistry showed The expression of hTERT protein in hAECs were negative.
Conclusions:
1.hAECs could be isolated successfully in vitro,but the cell only could be cultivated for short term in normal circumstances.
2.the hAECs were simple epithelium cells,hAECs had some characteristics of embryonic stem cells.
Part two Construction of eukaryotic high expression vector containing pLenti6/V5-DEST-hTERT-EGFP
Objective:
To construct eukaryotic high expressing vector containing pLenti6/V5-DEST-hTERT -EGFP gene using lentiviral vector,to establish experimental foundation for transfecting hAECs by pLenti6/V5-DEST -hTERT -EGFP.
Methods:
1 The construction and identification of gene-recombinated shuttle plasmid pD ONR221-hTERT-EGFP
Using the PCI-neo-hTERT plasmid as model,We obtained objective gene hTERT by PCR reactions.Through BP reaction,we directly cloned hTERT into pDONR221 to get pDONR221-hTERT.And using the pEGFP-N1 plasmid as model,We obtained production -linker-EGFP- through PCR reactions.Through ambi-enzyme-tomy and T4-DNA joining-enzyme reaction,the production attB1-hTERT-linker-EGFP-attB2 was obtained, then converted in Bacillus coli DH5α.The gene-recombinated shuttle plasmid vector pDONR221-hTERT-EGFP was identified by digestant,PCRreaction and sequencing.
2 The construction and identification of recombination carrier pLenti6/V5-DEST-hTERT-EGFP
Recombinant plasmid pLenti6/V5-DEST-hTERT-EGFP was obtained through LR recombinating reaction using recombinant plasmid vector pDONR221- hTERT-EGFP and carrier pLenti6/V5-DEST.Then,converted in Bacillus coli STB13 and cultured on LB flat plate containing ammine benzyl-and-mycin.The recombination carrier pLenti6/V5-DEST-hTERT-EGFP was identified by digestant,PCR reactions and sequencing.
3 The package and collection of supernatant containing hTERT-EGFP viral particle The 293FT cells were transfected with pLenti6/V5-DEST-hTERT-EGFP plasmid through lipofectctamine reagent,the supematant were collected by filtration method and conservated in—80℃.
Results:
1 The result of construction and identification of lentiviral rudiment carder pDONR221-hTERT -EGFP
Our study successfully amplificated attB1-hTERT-attB2 fragment which was as long as 3.4Kbp.hTERT directly cloned into lentiviral rudiment carder pDONR221 through BP reaction. The Results of sequencing and identification showed hTERT fragment had been successfully cloned into rudiment carrier pDONR221.
2 1547site rite-directed mutagenesis oflentivirus rudiment carrier pDONR221-hTERT-EGFP
Sequencing results indicated the objective gene hTERT of lentiviral rudiment carrier had mutanted in 1547 site.Then,we designed basi-primer containing correctiful 1547 site,the rite-directed mutagenesis of lentivirus rudiment carrier pDONR221-hTER fixed-point was succeeded and was confirmed by sequencing.
3 The result of the construction and identification of lentiviral recombinant carrier pLenti6/V5-DEST-hTERT-EGFP
lentiviral recombinant plasmid pLenti6/V5-DEST-hTERT-EGFP can be obtained through LR Recombinating reaction.The sequencing of recombinant plasmid confirmed that the construction of lentiviral recombinant plasmid pLenti6/V5-DEST -hTERT-EGFP was correct.
4 The result of supernatant containing hTERT-EGFP viral particle
The supernatant fluid cortaining hTERT-GFP viral particle were seccessfully collected through lipofectctamine reagent.
Conclusion:
1 We had successfully constructed lentiviral recombinant plasmid pLenti6/V5-DEST-hTERT-EGFP for the first time.
Part Three Transfaction and Expression of lentiviral rudiment carrier pLenti6/V5-DEST-hTERT-EGFP in hAECs
ⅠTransfer hAECs with pLenti6/V5-DEST-hTERT-EGFP
Objective:
To transfer hAECs with objective gene hTERT and marker gene EGFP by lentiviral vector, then amplify cells clone containing hTERT-EGFP gene.EGFP-positive rates and the change of transfected cell cycle were detected by flow cytometry examination,to establish experimental foundation for transfected cells transplantation in vivo.
Methods:
1 To culture human amniotic epithelium cells(hAECs) in vitro.
2 To transfer human amniotic epithelium cells(hAECs) with objective gene hTERT and marker gene EGFP by lentiviral vector.
2.1 Confirm the density of Blasticidin for filtrating.
We cultured hAECs with DMEM containing different level Blasticidin(0ug/ml、lug/ml、3ug/ml、5ug/ml、7ug/ml、10ug/ml) for about 14 days,observing the grow status of cells everday.
2.2 To transfer hAECs by Lentiviral vector
At the time of reaching 50%confluence of cells,hAECs were transfered with pLenti6/V5-DEST-hTERT-EGFP viral particle,the cells contained two orifices for experiment,two orifices for no-load control group,two orifices for normal control group, stoped transfection after 24h.the resistant clones secreting the recombinant proteins were estabilished with Blasticidin.
3 To observe the transient expression of transgene in the transfered hAECs with fluorescence microscope
we changed culture medium to DMEM without screening reagent and cultured cells sequentially after transfection.the expression of EGFP in hAECs was detected with fluorescence microscope respectively at the time of 12h、24h、48h、72h、96h after transfection
4 To culture extendly the cell clones
The cultured solution was changed with DMEM without screening reagent when the cell clones had appeared,then the cells were cultured for 1 week sequentially.the cells were trypsinized by trypsin/EDTA solution when the cells insoculated,then we extendly cultured the transfected hAECs..
5 To detect EGFP-positive rates of the transfered hAECs by flow cytometry examination
To collect the cells of nomal control hAECs and transient transfered hAECs groups of culturing 12h、24h、48h、72h、96h after transfection,then to examine marker gene EGFP in cells by flow cytometry and use cell-quest software to analyze EGFP-positive rates. 6 To examine the change of cell cycle by flow cytometry
To collect the hAECs cultured for 48h of stably transfected group、transfected empty plasmid group and normal control group with the density of 1×10~6 cells/ml,then to examine the cell cycle and calculate the proliferation index(PI) using flow cytometry.
Results:
1 The resistant clones were obtained by using DMEM containing 1ug/ml Blasticidin to culture for 10 days.
2 after cultured for 12h,the transient transfered hAECs could be observed the expression of green fluorescent protein in cytoplasm,but the amount was litter,the intensity was thin, only 0-2 positive cells could be seen in every low-power field.After 24h,the amount and intensity of green fluorescent protein gradually enhanced,about 2-5 positive cells could be seen in every low-power field.48h later,about over 10 positive cells could be seen in every low-power field,there were no difference between 48h and 72h.the amount and intensity of positive cells gradually decreased after 72h,2-3 positive cells could be seen in every low-power field at 96h.adding lug/ml Blasticidin and screening for 10d,mono cell clones appeared.
3 After transfected 2h,some transfected hAECs fell off.no obvious configuration difference have been found between transfered hAECs and normal hAECs.
4 EGFP-positive rate of transient transfected 48h group was the highest and was 18.53%.Compared with that of 12h,24h,96h groups,the difference had statistical significance(P＜0.05).Compared with that of 72h groups,the difference hadn't statistical significance(P＞0.05).Dense green fluorescence could been detected in the stably transfected groups.But EGFP-positive cells were not detected in the normal control group and transfected empty plasmid group.
5 The flow cytometry assay showed:Compared with normal control group,the cellular proportion of stabilly transfected hAECs group increased in S phase and G2/M phase significantly,and PI value increased,no distinct difference had been found between normal control group and no-load group.
Conclusions:
1 This is the first time for transfecting hAECs with objective gene hTERT and marker gene EGFP through Lentiviral vector 1,the transient expression and expression in transfected hAECs were all obtained.At the time of transfection 48h,the positive expression was highest and was 18.53%.
2 After transfected with pLenti6/V5-DEST-hTERT-EGFP through Lentiviral Vector,the PI of hAECs increased obviously,growth cycle of cells prolonged obviously too.
3 The results indicated the transfection through Lentiviral Vector had no influence to the cell appearance,differentiation and reproductive activity and other bionomics of hAECs,and showed that the Lentiviral Vector was a relative safeful gene transfection
ⅡThe expression of pLenti6/VS-DEST-hTERT-EGFP in transfered hAECs
Objective:
To detect the expression of hTERT mRNA gene and hTERT protein in transfected hAECs by Real Time fluorescent quantitation PCR and Western blotting.The immunohistochemical staining was used to detect hTERT protein expression in transfected hAECs,The athymic mouse experiment was used to detect the oncogenicity of stabilly transfected cells and to research the biological safety of transfected hAECs.
Methods:
1 To detect the expression of hTERT mRNA gene in transfected cells by Real Time fluorescent quantitation-PCR
1.1 The total RNA were abstracted from the cells cultured for 48h of normal control group,positive control group(HepG2 oncogenic cells),stably transfected group and transfected empty plasmid group with the density of 1×10~6 cells/ml.
1.2 The Purity and integrality of total RNA were detected by nucleic acid protein detection.the ratios of 00260/0D280 more than 1.8 showed RNA was fairly pure and no protein pollution.
1.3 The total RNA reverse- transcribed into cDNA by the PCR reactions.
1.4 After designing primer of qRT-PCR,then,begin to qRT-PCR reaction.
2 To detect the expression of hTERT Protein within the cells of four groups by using Westem blotting.
3 The immunohistoehemieal staining was used to detect expression of hTERT Protein within the transfected hAECs.
4 The athymic mouse experiment was used to detect the oncogenicity of stabilly transfected hAECs.
Results:
1 The results of Real Time fluorescent quantitation-PCR
The relative amount of hTERT mRNA in normal control group and no-load transfected control group are 0,the relative amount of positive control group(HepG2 oncogenic cells) is 450,stablly transfected group is 200,transient transfected group is 185,these results showed hTERT gene had been integrated in transfected hAECs on the level of mRNA and hTERT gene didn't express in normal hAECs and no-load transfected hAECs.
2 The results of Western blotting.
There was positive band of 314KD of hTERT protein in positive control group,not in normal control group,no-load transfected group and transfected group.
3 The results ofimmunohistochemical staining
The stablly transfected hAECs were stained to brown with immunohistochemical staining,but the cells of other groups were negative.
4 The results of athymic mouse experiment
After injected for two weeks,the mice injected HepG2 hepatoma carcinoma cells growed tumor in injection site and the tumor grew up along with the time,the mice injected transfected hAECs had't no change in the injection site.
Conclusions:
1 hTERT gene was confirmed to had integrated in gene of tablly transfected hAECs on levels of mRNA for the first time.
2 The stabilly transfected hAECs with objective gene hTERT have not been found oncogenicity at the time
Part Four Study on re-construction of corneal surface layer using transfered hAECs
Objective:
To study the subsistence of stabilly transfered hAECs on fresh corneal stroma.The fresh corneal stroma with transfered hAECs was transplanted onto the rabbit's corneal surface without limbal stem cells to reconstruct the corneal surface layer.To observe the survival of transfered h.AECs and the stability of ocular surface.GFP gene was used to detect the subsisitentce and distribution of transfered cells after transplantation in living cornea tissue.To establish experimental foundation for using stabilly transfected hAECs to re-establish the corneal surface layer.
Methods:
1 To creat Stem cell deficiency(SCD)models on 12 rabbits eyes
2 To culture transfered human amniotic epithelium cells(hAECs) in vitro.
3 To obtain fresh rabbit's corneal stroma
4 The transfered hAECs were seeded onto fresh corneal stroma surface.negative control group was seeded no cells.After cultured 10 days by Millicell culture plate inserts.The transfered hAECs and fresh corneal stroma fixed and dehydrated,observed by HE staining
4 The Stem cell deficiency(SCD) modols of New Zealand white rabbits were set up,and were divided into 2 groups:group A(normal control group,n=6):the stroma had not any cells,group B(pLenti6/V5-DEST-hTERT-EGFP group,n=6):the seed cells were hAECs transfered pLenti6/V5-DEST-hTERT-EGFP gene.
5 Corneal epithelium reconstruction procedures were performed after creating Stem cell deficiency(SCD)models for 2 months.During the surgery process,a 7.0mm diameter of corneal recipient bed was made.The corneal stroma with transfected cells or without any cells were placed on the recipient bed and sutured interruptedly using 10-0 suture.
6 The operated eyes were observed everyday since the first day after operation,the observation included corneal conjunctivalization,vascularization,and stromal opacity,and compared with each other for two months.
7 The structure of corneal implant in two groups were detected by HE attaining,the expression of of cytokeratin CK8、CK18、CK12 were detected by using immunohistochemical staining.
Results:
1 The transfered hAECs could grow well on the rabbit's corneal stromal cartier,after cultured on Millicell culture plate inserts for 10 days,the stabilly transfered hAECs were stratified and became corneal-like stratified epithelial cells by using the air-lifting cultivation.
2 during the observation period,six cornea of GroupA all became opaque and cloudy badlly.there were plentiful neovaseular around the grafts.five cornea of Group B maintained clear for 2 moulths,only one cornea rejected during observation Period.There were also a few neovaseular around the graft.
3 Imunohistochemistry staining showed:In group B,,the expression of of cytokeratin CK8、CK18、CK12 in epithelial lays were positive..In group A,Positive brown cells couldn't be observed in all lays.
Conclusions:
1 The function of corneal surface layer using transfered hAECs was more stable than control group.
2 The transfered hAECs with pLenti6/V5-DEST-hTERT-EGFP gene would be a new and excellent feed cells to reconstruct corneal surface layer.
第一部分人羊膜上皮细胞(hAECs)的体外培养和其生物学鉴定
目的:
研究人羊膜上皮细胞(hAECs)体外培养的生物学特性,探讨hAECs体外培养的方法及其干细胞特性,为基因转染hAECs并进行角膜表层重建奠定实验基础。
方法:
1采用酶消化法体外分离培养hAECs,观察细胞在体外培养条件下的生长情况,并对生长良好的原代细胞进行消化传代,在光镜下对细胞进行形态学观察和HE染色观察;
2对传代的hAECs进行细胞角蛋白CK7、CK8和CK18免疫组化染色鉴定及免疫荧光检测hAECs中干细胞转录因子OCT-4的表达;
3对传代的hAECs进行流式细胞仪检测干细胞表面分子标记CD29、CD34、CD44、CD45、CD105的表达。
4免疫组化检测传代hAECs中端粒酶催化亚基hTERT蛋白的表达。
结果:
1在体外培养条件下,hAECs能贴壁生长,细胞呈典型的上皮细胞样外观,正常情况下可传7—8代。HE染色可见细胞大小较均一,呈不规则的多角形,细胞连接成片。
2免疫组化结果显示培养的hAECs胞浆中见CK7、CK8、CK18单克隆抗体染色阳性,OCT-4免疫荧光结果显示hAECs的胞浆中有荧光表达。
3流式细胞仪检测发现hAECs干细胞标记分子CD29、CD34的表达为阳性,但干细胞标记分子CD44、CD45、CD105的表达为阴性。
4免疫组化结果显示培养的hAECs胞浆中hTERT单克隆抗体染色阴性。
结论:
1通过酶分离法可成功分离出纯度较高的hAECs,获得的细胞能在体外进行短期内培养。
2培养的hAECs是单层上皮细胞来源,并具有干细胞的某些特征。
第二部分真核表达载体pLenti6/V5-DEST-hTERT-EGFP的构建
目的:
利用慢病毒载体构建携带有目的基因人端粒酶催化亚基(hTERT)和标记基因增强型绿色荧光蛋白(EGFP)的真核表达载体pLenti6/V5-DEST-hTERT-EGFP,为基因转染hAECs奠定实验基础。
方法:
1基因重组穿梭质粒pDONR221-hTERT-EGFP的构建和鉴定
以PCI-neo-hTERT质粒为模板PCR法获取目的基因hTERT,通过BP反应,将hTERT定向克隆至pDONR221得pDONR221-hTERT;以质粒pEGFP-N1为模板,通过PCR反应得到-linker-EGFP-产物。通过双酶切,T4-DNA连接酶反应,得到pDONR221-hTERT-EGFP产物。然后转化大肠杆菌DH5α,卡那霉素筛选,挑克隆行PCR、酶切及测序鉴定。
2 pLenti6/V5-DEST-hTERT-EGFP重组载体的构建和鉴定
取pDONR221-hTERT-EGFP重组质粒和pLenti6/V5-DEST载体,进行LR重组反应,得到pLenti6/V5-DEST-hTERT-EGFP重组质粒,之后转化入大肠杆菌STB13感受态,涂含氨苄霉素的LB平板,挑取单菌落扩增培养,提取质粒,并进行测序鉴定。
3含hTERT-EGFP病毒颗粒上清的包装和收集
利用脂质体转染法将pLenti6/V5-DEST-hTERT-EGFP质粒转入293FT细胞,培养24h后,更换完全培养基,再48 h-72 h后,采用过滤法收集含病毒颗粒的上清液,一80℃保存备用。
结果:
1慢病毒入门载体pDONR221-hTERT-EGFP的构建和鉴定结果
本实验成功扩增出长达约3.4Kbp的attB1-hTERT-attB2片段,行PCR、酶切及测序鉴定,证实hTERT已克隆入入门载体pDONR221,并成功获得pDONR221-hTERT-EGFP产物。
2慢病毒入门载体pDONR221-hTERT的1547位点定点突变(将1547位点突变的碱基G变回原序列A)
测序结果表明慢病毒入门载体的目的基因hTERT存在1547位点发生错义突变,经设计含1547位点正确碱基的引物,通过定点突变技术将慢病毒入门载体pDONR221-hTERT的1547位点定点突变,经测序证实突变成功。
3慢病毒表达载体pLenti6/V5-DEST-hTERT-EGFP的构建和鉴定结果
经LR重组反应构建得慢病毒重组质粒pLenti6/V5-DEST-hTERT-EGFP,将重组质粒送去测序,证实重组质粒pLenti6/V5-DEST-hTERT-EGFP构建成功。
4含hTERT-GFP病毒颗粒上清液的制备结果
将pLenti6/V5-DEST-hTERT-GFP与Virapower~(TM)Packaging Mix利用脂质体共转染包装细胞293FT细胞,终止转染72h后收集上清液,过滤,即得含hTERT-GFP病毒颗粒的上清液。
结论:
1首次成功构建慢病毒重组质粒pLenti6/V5-DEST-hTERT-EGFP。
第三部分慢病毒表达载体pLenti6/V5-DEST-hTERT-EGFP的转染和表达
一、pLenti6/V5-DEST-hTERT-EGFP基因转染hAECs
目的:
在本实验中,利用慢病毒将目的基因人端粒酶逆转录酶基因(hTERT)和标记基因EGFP转染入人羊膜上皮细胞(hAECs),并扩增培养含hTERT-EGFP基因的克隆细胞,观察转染细胞的生长特性,用流式细胞仪检测转染细胞EGFP阳性表达率和检测hTERT基因转染对hAECs生长周期的影响,为下一步转染细胞的活体移植奠定实验基础。
方法:
1人羊膜上皮细胞的传代培养
2慢病毒载体的转染
2.1杀稻瘟菌素筛选浓度的确定
2.2慢病毒载体转染人羊膜上皮细胞
制成两孔转染实验组(转基因组)、两孔转空载对照组、两孔正常对照组,共六孔,24h后终止转染。
3筛选稳定表达的抗性细胞克隆并扩增培养
细胞终止转染后,继续培养24 h,加入含杀稻瘟菌素终浓度为1ug/ml的完全培养基,对细胞进行加压筛选,筛选10天左右,直至单个细胞克隆形成。重组细胞克隆形成后,换为不加筛选药物的DMEM完全培养基继续培养,至细胞接近融合时,消化传代,扩增培养。扩增培养时,仍加杀稻瘟菌素进行筛选。
4荧光显微镜下观察转染基因的瞬时表达
细胞终止转染后,用不加筛选药物的含10%胎牛血清的DMEM完全培养基继续培养12h、24h、48h、72h、96h,荧光显微镜下观察每个时间段瞬时转染细胞和稳定转染细胞绿色荧光蛋白的表达。
5倒置显微镜下观察转染细胞的生长形态
6流式细胞仪检测转染细胞EGFP阳性表达率
分别收集未转染的hAECs、瞬时转染12h、24h、48h、96h的hAECs,制成单细胞悬液,行流式细胞仪检测,利用cell-quesL软件分析EGFP阳性细胞表达率。
7流式细胞仪检测hTERT基因转染对hAECs生长周期的影响
分别收集稳定转染后第2代培养48h的hAECs(转基因组),常规传1代培养48h的hAECs(正常对照组),以及转染空载体传1代培养48h的hAECs(转染空载体组),调细胞浓度为1×10~6个/ml,用流式细胞仪检测标记细胞,CellQuest软件处理资料,全部数据采用SPSS11.0统计软件包进行处理,PI值比较采用单因素方差分析,以P＜0.05定为差异具有统计学意义。
结果:
1杀稻瘟菌素的抗性筛选
接种于6孔板中的人羊膜上皮细胞,当加入杀稻瘟菌素浓度为1ug/ml时,培养14d细胞全部死亡,从培养时间上考虑选1ug/ml作为杀稻瘟菌素的筛选浓度。
2倒置显微镜下所见
培养的人羊膜上皮细胞加入转染液12h后,有少数细胞从培养板上脱落下来漂浮于培养液中,大多数转染细胞筛选出的抗性克隆以及培养的克隆细胞均与正常细胞在形态上无显著差异。
3荧光显微镜下观察GFP的阳性表达
3.1瞬时转染12h后即可见发绿色荧光的人羊膜上皮细胞,但数量较少,强度教弱,每个低倍视野下可见0-2个;24h后发绿色荧光的人羊膜上皮细胞逐渐增多强度增强,每个低倍视野下可见5-8个左右;48h后发绿色荧光的人羊膜上皮细胞继续增多,每个低倍视野下可见10个以上;72h后发绿色荧光的人羊膜上皮细胞与48h的人羊膜上皮细胞差别不大;96h后发绿色荧光的人羊膜上皮细胞逐渐减少,每个低倍视野下可见5个左右,经1ug/ml的杀稻瘟菌素筛选10d后,观察有单细胞克隆出现。
3.2消化收集经1ug/ml的杀稻瘟菌素筛选获得稳定表达的人羊膜上皮细胞,接种于6孔板中扩增培养,贴壁后可见密集荧光。
4人羊膜上皮细胞pLenti6/V5-DEST-HTERT-EGFP基因转染效率检测结果流式细胞仪检测结果显示:未转染组人羊膜上皮细胞中,GFP阳性细胞数为零,瞬时转染12h、24h、48h、72h、96h组的人羊膜上皮细胞均可见GFP阳性表达,其中48h转染组的人羊膜上皮细胞GFP阳性表达率最高,为18.53%,与12h、24h、96h转染组比较差异具有统计学意义(P＜0.05),与72h转染组比较差异不显著,无统计学意义(P＞0.05)。
5细胞周期检测结果
转基因组人羊膜上皮细胞与正常对照组比较,处于S期和G2/M组的细胞比例增加,PI值升高,两者有显著性差异(P＜0.05),转空载体组人羊膜上皮细胞与正常对照组比较,PI值无显著变化(P＞0.05)。
结论:
1首次证明目的基因人端粒酶逆转录酶基因(hTERT)和标记基因EGFP经慢病毒介导转染入人羊膜上皮细胞(hAECs)后,并可在细胞内获得瞬时表达和稳定表达,且瞬时转染48h,人羊膜上皮细胞(hAECs)EGFP阳性表达率最高,为18.53%。
2转染入目的基因人端粒酶逆转录酶基因(hTERT)后,人羊膜上皮细胞(hAECs)的增值指数PI值明显升高,生长周期明显延长。
3慢病毒介导的转染方法对人羊膜上皮细胞(hAECs)的形态、分化和增殖能力等生物学特性无明显影响,再次证明是一种相对安全的基因转染方法。
二、pLenti6/V5-DEST-hTERT-EGFP基因在转基因hAECs中的表达
目的:
在本实验中,利用荧光定量PCR和Western blot方法从mRNA和蛋白质水平检测hTERT基因在转基因hAECs中的表达,并通过免疫组织化学法检测转基因hAECs中端粒酶催化亚基hTERT蛋白的表达,再通过裸鼠试验检测转染细胞的致瘤性,研究转染有hTERT基因的hAECs的生物安全性,为基因转染人羊膜上皮细胞并进行角膜表层重建奠定实验基础。
方法:
1应用荧光定量PCR(Real Time-PCR)方法检测hTERT mRNA在转染细胞中的表达
1.1总RNA提取(整个过程要求在超净台内进行)
分别提取常规传1代培养48h的hAECs(第1组,即正常对照组)、传1代培养48h的HepG2肝癌细胞(第2组,即阳性对照组)、以及稳定转染pLenti6/V5-DEST-hTERT-EGFP基因后第2代培养48h的hAECs(第3组,即稳定转染实验组)、瞬时转染48h的hAECs(第4组,即瞬时转染实验组)、用慢病毒空载体转染48h的hAECs(第5组,即空载转染对照组)的总RNA。
1.2总RNA纯度和完整性检测
1.2.1纯度检测:取双蒸水99μl加入比色杯中,再加入1μl总RNA样品,在核酸蛋白检测仪上测定OD值。
1.2.2总RNA完整性检测:取RNA样品2μl,1%琼脂糖凝胶电泳80V×30min,用凝胶成像系统观察总RNA的5s rRNA、18s rRNA和28s rRNA条带。
1.3逆转录:得1~(st) strand cDNA:
取上述5组细胞RNA液各2ul,转移到无菌PCR管中,行PCR反应。
1.4荧光定量qRT-PCR
1.4.1检测序列片段大小
1.4.2设计引物
1.4.3反应体系:2×2×SYBR green premix10μl+Primers mix0.8μl+cDNA1μl+H_2O 8.2μl,总体积为20μl;
1.4.4反应条件:95℃10min,95℃15s,65℃30s,72℃30s读板,共40个循环;
1.4.5融解曲线分析:温度55℃-95℃,每分钟读1次,每个样设置3个复孔。
2 Western blotting法检测转基因hAECs中端粒酶蛋白的表达
2.1样品的制备
取上述5组细胞,加入单去污剂裂解缓冲液,收集细胞裂解物,加入等体积凝胶加样缓冲液,离心后,将上清夜移至另一管中,待用。
2.2 SDS-聚丙烯酰胺凝胶电泳(SDS-PAGE)
2.2.1正确安装电泳槽玻璃板,加入分离胶,待分离胶完全聚合后,再在分离胶上灌注浓缩胶,快速插入干净的梳子,浓缩胶聚合后,小心移出梳子。
2.2.2将凝胶固定于电泳装置上,加入电泳缓冲液。按预定顺序加样,连接电泳装置与电流。当染料接近分离胶底部时,关闭电源。卸下玻璃板,分离玻璃板,标记凝胶方位。
2.3 Western Blotting法检测
电泳完毕后,将凝胶取下,贴于PVDF膜上,将胶、膜、滤纸、海绵和转移夹板夹成“三明治”状,进行转膜,闭膜,加一抗溶液孵育过夜。再加到二抗溶液中,摇床孵育1h。然后显色,感光,显影和定影,与之前预染的Marker对比,观察结果,拍照分析。
3免疫组化法检测转基因hAECs中端粒酶催化亚基hTERT蛋白的表达
采用链酶亲和素-过氧化物酶(SABC)法,检测盖玻片上常规培养的上述五种细胞中端粒酶催化亚基hTERT蛋白的表达情况。
4裸鼠致瘤实验
取稳定转染有目的基因hTERT的hAECs及HepG2肝癌细胞,取BALB/c—nu/nu品系裸鼠12只,6只接种转基因hAECs,6只接种HepG2肝癌细胞,于动物背部皮下无菌接种细胞悬液,屏障环境下饲养两周,观察细胞注射部位有无肿瘤生长以及肿瘤的大小。
结果:
1实时荧光定量PCR检测结果
第1组即正常对照组和第5组即空载转染对照组hTERTmRNA的相对含量为0即不表达hTERT,第2组即阳性对照组hTERT mRNA的相对含量为450,第3组即稳定转染组目的基因hTERT mRNA的相对含量为200,第4组即瞬时转染组目的基因hTERTmRNA的相对含量约为185,说明瞬时转染和稳定转染的hAECs内均有hTERTmRNA基因表达,而没有进行基因转染的hAECs和转染空载体的hAECs内无hTERTmRNA基因表达。
2 Western Blotting检测结果
第2组即阳性对照组细胞裂解产物,在相对分子量为314KD处呈现一条特异性蛋白条带,而第1组即正常对照组、和第3组即稳定转染组和第4组即瞬时转染组和第5组即空载转染对照组则无相应条带出现,这可能因为稳定转染组细胞中hTERT蛋白表达过少或没有表达。
3免疫组化检测结果
端粒酶催化亚基hTERT抗体在第1组即正常对照组、第4组即瞬时转染组和第5组即空载转染对照组的hAECs细胞胞浆中不着色,说明这些细胞不表达hTERT蛋白,而端粒酶催化亚基hTERT抗体在第2组即阳性对照组中细胞胞浆中呈深褐色着色,说明其hTERT蛋白表达呈强阳性,,TERT抗体在第3组即稳定转染组的hAECs中呈浅褐色着色,说明其hTERT蛋白表达呈弱阳性。
4裸鼠致瘤实验结果
裸鼠在屏障环境下饲养一周后,接种HepG2肝癌细胞的裸鼠注射部位即有肿瘤长出,并随着饲养天数的延长逐渐长大,而接种转基因hAECs的裸鼠注射部位无明显变化。
结论:
1首次从mRNA水平检测到hTERT基因在体外培养的hAECs中成功转染和表达,但蛋白质水平上没有检测到转染细胞中hTERT蛋白的表达,说明hTERT基因在mRNA水平和蛋白质水平的表达并不平衡。
2转染有目的基因hTERT且稳定表达的人羊膜上皮细胞目前尚未发现有致瘤性。
第四部分应用转基因hAECs重建角膜表层的研究
目的:
研究转基因人羊膜上皮细胞(hAECs)在新鲜角膜基质片上的生存情况,并将生长有转基因hAECs的新鲜角膜基质片移植到角膜缘干细胞缺乏的兔眼表面重建角膜表层,观察转基因人羊膜上皮细胞的存活情况及眼表的稳定性,探讨转基因HAECs在活体兔眼表面的转归,为基因转染hAECs并进行角膜表层重建奠定实验基础。
方法:
1制备兔角膜缘干细胞缺损模型
取实验兔12只,麻醉后,开睑器开睑,在兔右眼角膜表面滴用正庚醇数滴去除角膜上皮,环形切除角膜缘内2mm浅板层组织,剪去第3眼睑,结膜下注射庆大霉素及地塞米松,连续观察2个月,对角膜结膜化、新生血管情况进行记录。
2转基因pLenti6/V5-DEST-HTERT-EGFP人羊膜上皮细胞克隆的体外扩增培养
3新鲜兔角膜基质片的制备
取新西兰大白兔眼球,暴露角膜,吸取少量正庚醇,滴加到角膜表面去除角膜上皮层,留下光滑的角膜基质面。然后再在角膜缘处做一小板层切口,用无菌虹膜恢复器分离浅层角膜基质层。用7.5mm环钻钻取角膜基质,将其放在无菌超净台内风干,然后放入96孔板中,备用。
4在新鲜角膜基质片上种植转基因pLenti6/V5-DEST-HTERT-EGFP人羊膜上皮细胞并复层化
将转基因hAECs消化后,接种到96孔板中的新鲜角膜基质上,小心放入培养箱中静置培养。大约3天后,角膜基质片上的细胞形成单层融合时,取出基质片,放入事先放在6孔板内的带微孔滤膜、三维立体插入式细胞培养器中。置入细胞培养箱中静置培养,大约培养10天左右,自然形成上皮细胞的复层结构。
5新鲜角膜基质片上细胞的HE染色观察
新鲜角膜基质片上的细胞培养3天后和利用气液界面培养10天后,固定、脱水、包埋、切片,进行HE染色后光学显微镜观察。
6转基因hAECs移植片的眼表移植
6.1动物分组:将上述12只右眼角膜缘干细胞缺乏模型兔随机分成2组,按构建角膜移植片是否有细胞,分为:A组(对照组,n=6):新鲜角膜基质片上不种任何细胞,B组(pLenti6/V5-DEST-hTERT-EGFP基因转染组,n=6):种子细胞为含目的基因hTERT和标记基因EGFP的慢病毒载体转染的hAECs。
6.2模型兔麻醉后,开睑器开睑,用7.0mm环钻钻取兔右眼角膜中间组织,钝性分离及板层切除中央角膜组织,将转基因人羊膜上皮细胞移植片平铺于角膜创面,然后间断缝和移植片与角膜上。结膜下注射庆大霉素及地塞米松,局部涂四环素可的松眼膏,间断缝合上下睑缘。
7术后处置
术后术眼滴氧氟沙星滴眼液于结膜囊内,肌肉注射庆大霉素,并在饲料中加环胞霉素A。每日氧氟沙星滴眼液滴眼,每日观察植片的存活状况及眼表的稳定性。
8术后观察及检测
8.1大体观察
术后第一周内,每天在裂隙灯下观察,以后每周观察2-3次,连续观察2个月。对角膜水肿、混浊、结膜化、新生血管情况进行记录。
8.2移植眼角膜组织的HE染色观察
8.3移植眼角膜组织细胞角蛋白CK8、CK18、CK12免疫组化检测
结果:
1兔角膜缘干细胞缺乏模型
制备角膜缘干细胞缺乏模型术后观察,两个月后有10只兔眼表现为角膜表面结膜化,新生血管膜形成,全角膜灰白色混浊。另2只兔眼角膜没有出现明显的结膜化,但已长出大量新生血管,并逐渐向角膜中间蔓延。
2新鲜角膜基质片上细胞在倒置显微镜下所见
转基因hAECs接种到新鲜兔角膜基质片后,3h开始贴壁,12h细胞呈团块状堆积,24h后细胞呈棒状伸展,3d左右变成多角形,部分区域已连接成片。
3新鲜角膜基质片上细胞HE染色
转基因hAECs在新鲜兔角膜基质上3-5d左右形成致密的单层结构,细胞排列紧密,然后应用插入式细胞培养器形成的气.液界面培养法培养10d左右,转基因人羊膜上皮细胞能形成类似角膜上皮的3-5层复层结构,形成的复层上皮细胞与角膜基质连接紧密,不易脱落。
4.移植术后移植片的大体外观所见
A组:术后一个月观察,6只兔眼均表现为整个角膜高度水肿,角膜浑浊程度较重,有大量新生血管,角膜炎症反应重,分泌物多。术后两个月观察,角膜结膜化,角膜血管化严重,已向角膜中央侵犯。
B组:术后一个月观察,有6只兔眼角膜透明度有不同程度提高,角膜周边有新生血管,6只兔眼缝线已大部分脱落。术后两个月观察,5只兔眼角膜均恢复透明,角膜上皮光滑,只有1只兔眼角膜透明度较低,新生血管较多。
5.角膜移植片的HE染色所见
角膜上皮重建术后2月,B组角膜组织HE染色可见角膜表面上皮出现部分类似角膜上皮的复层排列,角膜基质层可见新生血管增生。
6.移植眼角膜组织免疫组化检测结果
角膜上皮重建术后2月,角膜组织免疫组化染色可见上皮细胞角蛋白CK8、CK18和角蛋白CK12均呈阳性表达,角膜表面上皮部分细胞胞浆呈棕黄色,细胞核蓝色。
结论:
1新鲜角膜基质有利于人羊膜上皮细胞形成类似角膜上皮的复层排列,且表达角膜上皮细胞特异性抗体CK12。
2转基因pLenti6/V5-DEST-hTERT-EGFP人羊膜上皮细胞可以成为重建角膜表层的一种新的种子细胞。
Engineering cornea epithelium surface layer need suitable seed cells.Researchers have found,Human amniotic epithelial cells(hAECs),with the characteristics of both embryonic stem cells and pluripotent stem cells,have the potential to differentiate into various tissue cells in particular condition.Being obtained easily and convenience,with no any ethical arguments,have much more chances to differentiate to corneal epithelial cells,So hAECs may be a new source of seed cells of tissue engineering cornea.But being terminally differentiated cells and with short duration in vitro,the hAECs were difficult to culture.This shortcoming restricted its application in cell therapy.In view of this,us used this fusion gene of destination gene hTERT and marker gene amplification green fluorescent protein(GFP) to transfect hAECs,which compositive with lentivirus vector,to prolong cells growth cycle,even immortalized them.next,utilizing this trans-gened hAECs to re-construct laminated epithelium and cornea surface layer,to investigate application value of the trans-gened hAECs as a new seed cell souce of engineering cornea epithelium surface layer.
Part One The culture and biologic identification of human amniotic epithelial cells(hAECs) in vitro
Objective:
To study the biologic characteristics of the cultured human amniotic epithelial cells(hAECs) in vitro,and to investigate the culturing method and stem cell characteristics of hAECs;to establish experimental foundation for transfecting hAECs with hTERTgene and reconstructing corneal surface layer.
Methods:
1.We used enzyme digestion to isolate and culture hAECs and observed the adherence and growth of the cells,the morphologic changes of cultured AECs were observed by HE staining,inverted microscope and light microscope.
2.The expression of cytokeratin CK7、CK8 and CK18 in AECs were detected by using immunohistochemical staining.The immunofluorescence was used to detect the expression of OCT-4 of stem cell factor in passaged hAECs.
3.Stem cell surface molecule marker CD29、CD34、CD44、CD45、CD 105 were detected by flow cytometry in passaged hAECs.
4.Using immunohistochemistry to study the expression of hTERT protein in passaged hAECs
Results:
1 hAECs could adhere and grow very well in vitro,the cells looked like slabstone, they could passage 7—8 generations in normal circumstances.
2 The results of immunohistochemistry showed The hAECs expressed cytokeratin CK7、CK8、CK18 in cytoplasm,the result of immunofluorescence showed The expression of OCT-4 of stem cell factor in hAECs was positive too.
3 Detection of stem cell surface molecule marker by the flow cytometry showed The expression of CD29、CD34 on hAECs were positive,The expression of CD44、CD45、CD 105 on hAECs were negative.
4 The results of immunohistochemistry showed The expression of hTERT protein in hAECs were negative.
Conclusions:
1.hAECs could be isolated successfully in vitro,but the cell only could be cultivated for short term in normal circumstances.
2.the hAECs were simple epithelium cells,hAECs had some characteristics of embryonic stem cells.
Part two Construction of eukaryotic high expression vector containing pLenti6/V5-DEST-hTERT-EGFP
Objective:
To construct eukaryotic high expressing vector containing pLenti6/V5-DEST-hTERT -EGFP gene using lentiviral vector,to establish experimental foundation for transfecting hAECs by pLenti6/V5-DEST -hTERT -EGFP.
Methods:
1 The construction and identification of gene-recombinated shuttle plasmid pD ONR221-hTERT-EGFP
Using the PCI-neo-hTERT plasmid as model,We obtained objective gene hTERT by PCR reactions.Through BP reaction,we directly cloned hTERT into pDONR221 to get pDONR221-hTERT.And using the pEGFP-N1 plasmid as model,We obtained production -linker-EGFP- through PCR reactions.Through ambi-enzyme-tomy and T4-DNA joining-enzyme reaction,the production attB1-hTERT-linker-EGFP-attB2 was obtained, then converted in Bacillus coli DH5α.The gene-recombinated shuttle plasmid vector pDONR221-hTERT-EGFP was identified by digestant,PCRreaction and sequencing.
2 The construction and identification of recombination carrier pLenti6/V5-DEST-hTERT-EGFP
Recombinant plasmid pLenti6/V5-DEST-hTERT-EGFP was obtained through LR recombinating reaction using recombinant plasmid vector pDONR221- hTERT-EGFP and carrier pLenti6/V5-DEST.Then,converted in Bacillus coli STB13 and cultured on LB flat plate containing ammine benzyl-and-mycin.The recombination carrier pLenti6/V5-DEST-hTERT-EGFP was identified by digestant,PCR reactions and sequencing.
3 The package and collection of supernatant containing hTERT-EGFP viral particle The 293FT cells were transfected with pLenti6/V5-DEST-hTERT-EGFP plasmid through lipofectctamine reagent,the supematant were collected by filtration method and conservated in—80℃.
Results:
1 The result of construction and identification of lentiviral rudiment carder pDONR221-hTERT -EGFP
Our study successfully amplificated attB1-hTERT-attB2 fragment which was as long as 3.4Kbp.hTERT directly cloned into lentiviral rudiment carder pDONR221 through BP reaction. The Results of sequencing and identification showed hTERT fragment had been successfully cloned into rudiment carrier pDONR221.
2 1547site rite-directed mutagenesis oflentivirus rudiment carrier pDONR221-hTERT-EGFP
Sequencing results indicated the objective gene hTERT of lentiviral rudiment carrier had mutanted in 1547 site.Then,we designed basi-primer containing correctiful 1547 site,the rite-directed mutagenesis of lentivirus rudiment carrier pDONR221-hTER fixed-point was succeeded and was confirmed by sequencing.
3 The result of the construction and identification of lentiviral recombinant carrier pLenti6/V5-DEST-hTERT-EGFP
lentiviral recombinant plasmid pLenti6/V5-DEST-hTERT-EGFP can be obtained through LR Recombinating reaction.The sequencing of recombinant plasmid confirmed that the construction of lentiviral recombinant plasmid pLenti6/V5-DEST -hTERT-EGFP was correct.
4 The result of supernatant containing hTERT-EGFP viral particle
The supernatant fluid cortaining hTERT-GFP viral particle were seccessfully collected through lipofectctamine reagent.
Conclusion:
1 We had successfully constructed lentiviral recombinant plasmid pLenti6/V5-DEST-hTERT-EGFP for the first time.
Part Three Transfaction and Expression of lentiviral rudiment carrier pLenti6/V5-DEST-hTERT-EGFP in hAECs
ⅠTransfer hAECs with pLenti6/V5-DEST-hTERT-EGFP
Objective:
To transfer hAECs with objective gene hTERT and marker gene EGFP by lentiviral vector, then amplify cells clone containing hTERT-EGFP gene.EGFP-positive rates and the change of transfected cell cycle were detected by flow cytometry examination,to establish experimental foundation for transfected cells transplantation in vivo.
Methods:
1 To culture human amniotic epithelium cells(hAECs) in vitro.
2 To transfer human amniotic epithelium cells(hAECs) with objective gene hTERT and marker gene EGFP by lentiviral vector.
2.1 Confirm the density of Blasticidin for filtrating.
We cultured hAECs with DMEM containing different level Blasticidin(0ug/ml、lug/ml、3ug/ml、5ug/ml、7ug/ml、10ug/ml) for about 14 days,observing the grow status of cells everday.
2.2 To transfer hAECs by Lentiviral vector
At the time of reaching 50%confluence of cells,hAECs were transfered with pLenti6/V5-DEST-hTERT-EGFP viral particle,the cells contained two orifices for experiment,two orifices for no-load control group,two orifices for normal control group, stoped transfection after 24h.the resistant clones secreting the recombinant proteins were estabilished with Blasticidin.
3 To observe the transient expression of transgene in the transfered hAECs with fluorescence microscope
we changed culture medium to DMEM without screening reagent and cultured cells sequentially after transfection.the expression of EGFP in hAECs was detected with fluorescence microscope respectively at the time of 12h、24h、48h、72h、96h after transfection
4 To culture extendly the cell clones
The cultured solution was changed with DMEM without screening reagent when the cell clones had appeared,then the cells were cultured for 1 week sequentially.the cells were trypsinized by trypsin/EDTA solution when the cells insoculated,then we extendly cultured the transfected hAECs..
5 To detect EGFP-positive rates of the transfered hAECs by flow cytometry examination
To collect the cells of nomal control hAECs and transient transfered hAECs groups of culturing 12h、24h、48h、72h、96h after transfection,then to examine marker gene EGFP in cells by flow cytometry and use cell-quest software to analyze EGFP-positive rates. 6 To examine the change of cell cycle by flow cytometry
To collect the hAECs cultured for 48h of stably transfected group、transfected empty plasmid group and normal control group with the density of 1×10~6 cells/ml,then to examine the cell cycle and calculate the proliferation index(PI) using flow cytometry.
Results:
1 The resistant clones were obtained by using DMEM containing 1ug/ml Blasticidin to culture for 10 days.
2 after cultured for 12h,the transient transfered hAECs could be observed the expression of green fluorescent protein in cytoplasm,but the amount was litter,the intensity was thin, only 0-2 positive cells could be seen in every low-power field.After 24h,the amount and intensity of green fluorescent protein gradually enhanced,about 2-5 positive cells could be seen in every low-power field.48h later,about over 10 positive cells could be seen in every low-power field,there were no difference between 48h and 72h.the amount and intensity of positive cells gradually decreased after 72h,2-3 positive cells could be seen in every low-power field at 96h.adding lug/ml Blasticidin and screening for 10d,mono cell clones appeared.
3 After transfected 2h,some transfected hAECs fell off.no obvious configuration difference have been found between transfered hAECs and normal hAECs.
4 EGFP-positive rate of transient transfected 48h group was the highest and was 18.53%.Compared with that of 12h,24h,96h groups,the difference had statistical significance(P＜0.05).Compared with that of 72h groups,the difference hadn't statistical significance(P＞0.05).Dense green fluorescence could been detected in the stably transfected groups.But EGFP-positive cells were not detected in the normal control group and transfected empty plasmid group.
5 The flow cytometry assay showed:Compared with normal control group,the cellular proportion of stabilly transfected hAECs group increased in S phase and G2/M phase significantly,and PI value increased,no distinct difference had been found between normal control group and no-load group.
Conclusions:
1 This is the first time for transfecting hAECs with objective gene hTERT and marker gene EGFP through Lentiviral vector 1,the transient expression and expression in transfected hAECs were all obtained.At the time of transfection 48h,the positive expression was highest and was 18.53%.
2 After transfected with pLenti6/V5-DEST-hTERT-EGFP through Lentiviral Vector,the PI of hAECs increased obviously,growth cycle of cells prolonged obviously too.
3 The results indicated the transfection through Lentiviral Vector had no influence to the cell appearance,differentiation and reproductive activity and other bionomics of hAECs,and showed that the Lentiviral Vector was a relative safeful gene transfection
ⅡThe expression of pLenti6/VS-DEST-hTERT-EGFP in transfered hAECs
Objective:
To detect the expression of hTERT mRNA gene and hTERT protein in transfected hAECs by Real Time fluorescent quantitation PCR and Western blotting.The immunohistochemical staining was used to detect hTERT protein expression in transfected hAECs,The athymic mouse experiment was used to detect the oncogenicity of stabilly transfected cells and to research the biological safety of transfected hAECs.
Methods:
1 To detect the expression of hTERT mRNA gene in transfected cells by Real Time fluorescent quantitation-PCR
1.1 The total RNA were abstracted from the cells cultured for 48h of normal control group,positive control group(HepG2 oncogenic cells),stably transfected group and transfected empty plasmid group with the density of 1×10~6 cells/ml.
1.2 The Purity and integrality of total RNA were detected by nucleic acid protein detection.the ratios of 00260/0D280 more than 1.8 showed RNA was fairly pure and no protein pollution.
1.3 The total RNA reverse- transcribed into cDNA by the PCR reactions.
1.4 After designing primer of qRT-PCR,then,begin to qRT-PCR reaction.
2 To detect the expression of hTERT Protein within the cells of four groups by using Westem blotting.
3 The immunohistoehemieal staining was used to detect expression of hTERT Protein within the transfected hAECs.
4 The athymic mouse experiment was used to detect the oncogenicity of stabilly transfected hAECs.
Results:
1 The results of Real Time fluorescent quantitation-PCR
The relative amount of hTERT mRNA in normal control group and no-load transfected control group are 0,the relative amount of positive control group(HepG2 oncogenic cells) is 450,stablly transfected group is 200,transient transfected group is 185,these results showed hTERT gene had been integrated in transfected hAECs on the level of mRNA and hTERT gene didn't express in normal hAECs and no-load transfected hAECs.
2 The results of Western blotting.
There was positive band of 314KD of hTERT protein in positive control group,not in normal control group,no-load transfected group and transfected group.
3 The results ofimmunohistochemical staining
The stablly transfected hAECs were stained to brown with immunohistochemical staining,but the cells of other groups were negative.
4 The results of athymic mouse experiment
After injected for two weeks,the mice injected HepG2 hepatoma carcinoma cells growed tumor in injection site and the tumor grew up along with the time,the mice injected transfected hAECs had't no change in the injection site.
Conclusions:
1 hTERT gene was confirmed to had integrated in gene of tablly transfected hAECs on levels of mRNA for the first time.
2 The stabilly transfected hAECs with objective gene hTERT have not been found oncogenicity at the time
Part Four Study on re-construction of corneal surface layer using transfered hAECs
Objective:
To study the subsistence of stabilly transfered hAECs on fresh corneal stroma.The fresh corneal stroma with transfered hAECs was transplanted onto the rabbit's corneal surface without limbal stem cells to reconstruct the corneal surface layer.To observe the survival of transfered h.AECs and the stability of ocular surface.GFP gene was used to detect the subsisitentce and distribution of transfered cells after transplantation in living cornea tissue.To establish experimental foundation for using stabilly transfected hAECs to re-establish the corneal surface layer.
Methods:
1 To creat Stem cell deficiency(SCD)models on 12 rabbits eyes
2 To culture transfered human amniotic epithelium cells(hAECs) in vitro.
3 To obtain fresh rabbit's corneal stroma
4 The transfered hAECs were seeded onto fresh corneal stroma surface.negative control group was seeded no cells.After cultured 10 days by Millicell culture plate inserts.The transfered hAECs and fresh corneal stroma fixed and dehydrated,observed by HE staining
4 The Stem cell deficiency(SCD) modols of New Zealand white rabbits were set up,and were divided into 2 groups:group A(normal control group,n=6):the stroma had not any cells,group B(pLenti6/V5-DEST-hTERT-EGFP group,n=6):the seed cells were hAECs transfered pLenti6/V5-DEST-hTERT-EGFP gene.
5 Corneal epithelium reconstruction procedures were performed after creating Stem cell deficiency(SCD)models for 2 months.During the surgery process,a 7.0mm diameter of corneal recipient bed was made.The corneal stroma with transfected cells or without any cells were placed on the recipient bed and sutured interruptedly using 10-0 suture.
6 The operated eyes were observed everyday since the first day after operation,the observation included corneal conjunctivalization,vascularization,and stromal opacity,and compared with each other for two months.
7 The structure of corneal implant in two groups were detected by HE attaining,the expression of of cytokeratin CK8、CK18、CK12 were detected by using immunohistochemical staining.
Results:
1 The transfered hAECs could grow well on the rabbit's corneal stromal cartier,after cultured on Millicell culture plate inserts for 10 days,the stabilly transfered hAECs were stratified and became corneal-like stratified epithelial cells by using the air-lifting cultivation.
2 during the observation period,six cornea of GroupA all became opaque and cloudy badlly.there were plentiful neovaseular around the grafts.five cornea of Group B maintained clear for 2 moulths,only one cornea rejected during observation Period.There were also a few neovaseular around the graft.
3 Imunohistochemistry staining showed:In group B,,the expression of of cytokeratin CK8、CK18、CK12 in epithelial lays were positive..In group A,Positive brown cells couldn't be observed in all lays.
Conclusions:
1 The function of corneal surface layer using transfered hAECs was more stable than control group.
2 The transfered hAECs with pLenti6/V5-DEST-hTERT-EGFP gene would be a new and excellent feed cells to reconstruct corneal surface layer.
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