肝细胞生长因子基因转染脂肪细胞对移植组织工程脂肪存活的影响**★

背景：在脂肪组织工程的研究中，若将种子细胞与支架材料复合后移植，则此组织工程脂肪将可替代颗粒脂肪。肝细胞生长因子可以促进血管生成、减少纤维组织增生。 目的：观察肝细胞生长因子基因修饰骨髓间充质干细胞来源的脂肪细胞对大鼠组织工程脂肪移植存活的影响。 设计、时间及地点：随机区组设计的动物实验，于2006-08/2007-07在解放军兰州军区兰州总医院医学科学实验中心完成。 材料：纳入Wistar大鼠用于分离、培养骨髓间充质干细胞及植入细胞-支架复合物。 方法：①分离培养雄性大鼠骨髓间充质干细胞，诱导成脂，携带绿色荧光蛋白基因及肝细胞生长因子基因的重组腺病毒毒株分别转染脂肪细胞，流式细胞仪测定转染效率，应用酶联免疫吸附试验法测定培养上清中肝细胞生长因子的表达，将脂肪细胞接种于聚乳酸聚乙醇酸共聚物支架。②将75只Wistar雌性大鼠按随机数字表法分为3组：脂肪细胞组、Ad-肝细胞生长因子转染组及Ad-绿色荧光蛋白转染组分别将脂肪细胞-聚乳酸聚乙醇酸共聚物、转染肝细胞生长因子基因脂肪细胞-聚乳酸聚乙醇酸共聚物、转染绿色荧光蛋白基因脂肪细胞-聚乳酸聚乙醇酸共聚物移植于大鼠腿部肌肉之间。 主要观察指标：移植后3，7，14，28，60 d，每组各取5只大鼠麻醉后处死，取出移植物。电镜观察；并行苏木精-伊红、油红O、Masson’s染色，观察病理改变；同时用免疫组织化学法检测组织中肝细胞生长因子表达，观察并计数血管生成。 结果：①骨髓间充质干细胞来源的脂肪细胞胞浆内富含丰富的脂滴，重组腺病毒感染复数=100时，携带绿色荧光蛋白基因的重组腺病毒株转染脂肪细胞效率可达64.39%。②移植3，7，14 d时，Ad-肝细胞生长因子转染组移植物中肝细胞生长因子的表达高于其他组（P < 0.05），且3 d达到高峰。③移植3，7，14，28，60 d时，Ad-肝细胞生长因子转染组移植物中血管生成高于其他组（P < 0.05），且7 d时血管增加的幅度最大。④移植28，60 d时，Ad-肝细胞生长因子转染组坏死程度均小于其他组，电镜下脂肪细胞也多于其他组。 结论：携带肝细胞生长因子基因的脂肪细胞在移植物中能够表达肝细胞生长因子，促进移植组织工程脂肪血管生成，进而促进移植组织的成活。

Effect of adipose cells transfected with recombinant adenovirus carrying human hepatocyte growth factor gene on survival of tissue-engineered adipose grafts

BACKGROUND: In adipose tissue engineering, seed cell and scaffold composite can replace particle adipose. Hepatocyte growth factor (HGF) can promote angiogenesis and reduce fibroplasia. OBJECTIVE: To study the effect of adipose cells transfected with recombinant adenovirus carrying human HGF gene on survival of tissue-engineered adipose grafts. DESIGN, TIME AND SETTING: Randomized animal trial was performed at the Experimental Center of Medical Science, Lanzhou General Hospital of Lanzhou Area Command of Chinese PLA. MATERIALS: Wistar rats were selected to isolate and culture bone marrow mesenchymal stem cells (MSCs). METHODS: MSCs of male Wistar rats were collected and cultured in vitro, to induce to differentiate into adipose cells. The adipose cells were transfected with Ad-GFP or Ad-HGF. The transfection infectivity was assayed by fluorescent microsopy and flow cytometry, and the expression of HGF was measured using ELISA assay. The adipose cells were seeded into poly(lactic-co-glycolic acid) (PLGA) scaffold. Seventy-five female rats were randomly divided into three groups (n=25): adipose cells-PLGA, adipose cells transfected Ad-HGF-PLGA, and adipose cells transfected Ad-GFP-PLGA groups. Then they were transplanted between the muscles of the rat legs. MAIN OUTCOME MEASURES: Adipose grafts were obtained on days 3, 5, 7, 14, 28, and 60. Absorption of adipose graft and collagen proliferation were examined by electron microscope and pathology staining. The expression of HGF and CD34 in transplanted adipose tissue was detected by immunohistochemistry. RESULTS: Abundant lipid droplet was observed in MSC-derived adipose cells. The transfection infectivity of Ad-GFP to adipose cells was 64.39% at 100 MOI. The expression of HGF in adipose cells culture medium of adipose cells transfected Ad-HGF-PLGA was higher than the other groups (P < 0.05), and reached to the peak level on day 3. Microvessel quantity in adipose cells transfected Ad-HGF-PLGA group was much more than that in the other groups on days 3, 7, 14, 28, and 60 post-transplantation (P < 0.05), especially on day 7. On days 28 and 60 post-transplantation, cell necrosis degree in adipose cells transfected Ad-HGF-PLGA group was more slightly than the other groups, and number of adipose cells was more than the other groups. CONCLUSION: The adipose cells transfected with Ad-HGF can secrete HGF, and promote angiogenesis and survival of adipose grafts.