【作者】 程昌志；

【导师】 张正治；

【作者基本信息】 第三军医大学 ， 人体解剖学， 2003， 硕士

【摘要】 多项研究证实，重组的人类胰岛素样生长因子-1（rhIGF-1）对肌腱细胞有促进分裂增殖的作用，IGF-1还能以剂量依赖型的方式明显促进胶原、蛋白多糖等细胞外基质的生成。但是rhIGF-1存在半衰期短、给药不方便等缺点，这些都限制了IGF-1的应用。采用基因转染技术，将外源基因定向导入肌腱组织并恒定表达，将有望充分发挥IGF-1的促分裂增殖作用。本课题取成年幼鼠后肢屈趾肌腱以改良的组织块法培养原代肌腱细胞并传代，将质粒pAdlox-IGF-1和pcDNA3.1(+)转化DH5α并提取质粒，用限制酶Hind III和BamHⅠ双酶切并回收前者的短片段和后者长片段，T4DNA连接酶体外连接，转化并提取连接产物，酶切及测序鉴定，即可得到IGF-1cDNA的真核表达载体pcDNA3.1(+)－IGF-1。并将pcDNA3.1(+)-IGF-1以脂多胺DOGS介导转染离体培养的大鼠屈趾肌腱细胞，进行短暂表达研究。转染后48小时提取转染细胞总RNA，以RT-PCR鉴定IGF-1mRNA的表达；并收集培养上清，检测IGF-1活性蛋白的表达情况。为了观察外源性IGF-1对肌腱细胞分裂增殖的促进作用，本实验用3H-TdR掺入法检测肌腱细胞DNA的合成情况：转染前24小时，以2×105/ml的浓度接种细胞于96孔培养板，每孔150 μl；然后分别于转染后24小时、48小时和72小时加入3H-TdR（每孔1 μCi）共培养6小时后收集细胞，检测3H-TdR掺入的CPM值。以空质粒转染组为对照。同时进行细胞的爬片培养，行Ⅰ型胶原的免疫荧光及激光共聚焦显微镜检测Ⅰ型胶原的表达。实验结果如下：1．成功构建真核表达载体pcDNA3.1(+)－IGF-1经酶切、回收及体外连接，获得质粒pcDNA3.1(+)－IGF-1，经酶切及测序鉴定，证实真核表达载体pcDNA3.1(+)－IGF-1构建成功。2．原代肌腱细胞培养及传代原代培养的大鼠肌腱细胞呈长梭形、三角形、多角形等多种形态，初始阶段以长梭形居多，随着培养时间增加，形态变为以多角形居多。并且形体逐渐变大，胞核明显且较大。传代培养细胞至第三代，生长能力逐渐变弱，传代的细胞很快老化。Ⅰ、Ⅲ型胶原的免疫细胞化学染色显示Ⅰ型胶原染色强阳性，Ⅲ型胶原染色阴性或弱阳性，说明培养的细胞确实为肌腱细胞。3．IGF-1mRNA的表达<WP=8>经过RT-PCR反应，扩增产物经1.5%琼脂糖凝胶电泳证实转染组成功扩增出目的片段(365 bp)和β－actin(441 bp)，而对照组RNA则未扩增出目的片段，证明肌腱细胞正确表达了IGF-1mRNA。4．IGF-1蛋白的表达IGF-1的 ELISA试剂盒检测显示，转染组上清IGF-1活性蛋白的表达量较对照组明显增高(P<0.05)，说明IGF-1活性蛋白已顺利表达，转染成功。5．肌腱细胞3H-TdR的掺入3H-TdR掺入法检测结果显示转染组和对照组的CPM值峰值均出现在第二时段（转染后48小时），实验组CPM值为2355.75±541.98(±s)，而对照组CPM值为1149.00±485.30(±s)，前者较后者明显增高(P<0.05)。说明IGF-1的转染明显促进肌腱细胞DNA的合成。而另外两个时段实验组和对照组的CPM值均很低，而且两组间没有显著差异，说明肌腱细胞可能处于非S期。6．IGF-1转染对肌腱细胞Ⅰ型胶原的影响细胞爬片经免疫荧光染色及激光共聚焦检测，结果显示转染组细胞和对照组细胞Ⅰ型胶原染色均为阳性，转染组荧光强度为76.20±32.23(±s， n=12)，而对照组为38.84±11.10(±s， n=10)，转染组较对照组表达明显增强(P<0.01)。以上实验结果表明，IGF-1基因能够通过真核表达载体pcDNA3.1(+)－IGF-1成功转染肌腱细胞并顺利表达，IGF-1的表达能够促进肌腱细胞的生长和Ⅰ型胶原的分泌，可能对肌腱细胞的分裂生长有明显促进作用。揭示了生长因子在肌腱损伤后的转基因治疗中将有良好的应用前景。更多还原

【Abstract】 It has been shown that recombinant human insulin-like growth factor 1(rhIGF-1) could stimulate tendon cells to proliferation and increase the production of collagen and proteoglycan in a dose-dependent manner. And promote flexor tendon healing in vivo. However, the short half-life of rhIGF-1 in vivo and lack of suitable stable vehicle for IGF-1 administration limited its potential application in tendon repair. Gene mediated directional transfection of tendon tissue will be a good way to fullfill the function of IGF-1. Primary rat flexor tendon cells were cultured by improving pieces culture technique and passaged successfully. The plasmids pAdlox-IGF-1 and pcDNA3.1(+) were transformed and then extracted from the competent DH5α strain. After digested by Hind III and BamHⅠ，the short fragment of pAdlox-IGF-1 and the long fragment of pcDNA3.1(+) were recovered and linked to construct pcDNA3.1(+)-IGF-1 with T4 DNA ligase in vitro. The vector was transferd to the secondary passage rat tendon cells mediated by lipopolyamine DOGS. 48 hours after the transfection, the total RNA and culture medium of tendon cells were collected for measurement of mRNA by RT-PCR and IGF-1 active protein by enzyme-linked immunoabsorbent assay(ELISA) respectively. To identify the influence of IGF-1 active protein on tendon cells, 3H-TdR incorporation and collagen type Ⅰ production of tendon cells were detected. The tendon cells which density was 2×105/ml were passaged to 96 well plate as 150μl per well. After 24 hours, 48 hours and 72 hours of the transfection, 1 μCi 3H-TdR per well was added to the cells and cultured continuously for six hours, then the cells were collected to assay the incorporation of the 3H-TdR respectively. Null vector transfection as the control. Moreover, both of the transfected tendon cells and the untransfected cells were passaged to grow on small slide for immunofluoresence of collagen typeⅠ. The results are below:1. Eukaryotic expression vector of pcDNA3.1(+)-IGF-1 was constructed successfully By digestion, recovery and ligation in vitro, the recombined eukaryotic expression vector pcDNA3.1(+)-IGF-1 was constructed and identified by digestion with the same two restriction endonuclease and sequencing with T7 promotor primer. Both of the results show that the vector was successfully constructed.<WP=5>2. Primary rat flexor tendon cells were cultured and passaged successfullyPrimary cultured rat flexor tendon cells were fusiform shape, deltoid and polygonal. In early days more of them were long fusiform shape. And finally most of them became polygonal and the antennas were very long and connect to each other. After passaging two generations, the cells began to aging and grow more and more weaker. Immunocytochemistry of collagen type Ⅰand type Ⅲ show that collagen type Ⅰ staining was very stronger than that of collagen type Ⅲ. It proved that the cultured cells were tendon cells. 3. The expression of IGF-1mRNAThe electrophoresis results of 1.5% sepharose of RT-PCR products show that there were two fragments that had been amplifyied in the transfected group. One is IGF-1mRNA(365 bp) and the other isβ-actin(441 bp). However, there was only oneβ-actin(441 bp) fragment found in the control group. The results manifested that IGF-1mRNA has expressed successfully in the transfected tendon cells. 4. The expression of IGF-1 biological active proteinEnzyme-linked immunoabsorbent assay(ELISA) of insulin-like grouth factor 1(IGF-1) of the cultured medium show that the content of IGF-1 of the experimental group was 111.71±18.60 ng/ml(±s), which was significant higher than that of the control 74.23±25.99 ng/ml(±s) (P﹤0.05). 5. 3H-TdR incorporation of the tendon cellsSix hours after adding the 3H-TdR to the tendon cells, 3H-TdR incorporation of the cells was assaied by 3H-TdR method. The results show that both of the experimental group and control group’s incorporation peak of 3H-TdR occur at 48 hours after the transfection. The CPM of the transfected group was 2355.75±541.98(?更多还原