Lei Lei, Liao Weiming, Sheng Puyi, Huang Gang, Jiang Li
Department of Orthopaedics, 1st Affiliated Hospital, Sun Yat-Sen University, Guangzhou Guangdong 510080, P R China.
Zhongguo Xiu Fu Chong Jian Wai Ke Za Zhi. 2006 Feb;20(2):134-8.
To explore an experimental method of transfecting the marrow stromal stem cells (MSCs) with the reconstructed PGL3-transforming growth factor-beta1 (TGF-beta1) gene and to evaluate the feasibility of self-induction of MSCs to the chondrocytes in vitro so as to provide a scientific and experimental basis for a further "gene enhanced tissue engineering" research.
The rabbit MSCs was transfected with the reconstructed PGL3-TGF-beta1, gene by the Liposomes Method, the growth of the cells were observed, and the growth curve was drawn. The living activity of the transfected cells in the experimental group was evaluated by MTT, and the result was significantly different when compared with that in the control group. By the immunohistochemistry method (SABC), the antigens of TGF-beta1 and collagen II were examined at 2 and 7 days of the cell culture after transfection with PGL3-TGF-beta1 gene. The pictures of the immunohistochemistry slice were analyzed with the analysis instrument, and the statistical analysis was performed with the software of the SPSS 11.0, compared with the control group and the blank group.
Transfection of the cultured rabbit MSCs in vitro with the reconstructed PGL3-TGF-beta1 gene by the Liposomes Method achieved a success, with a detection of the Luceraferase activity. The result was significantly different from that in the control group (P < 0.01). Tested by MTT, the living activity of the transfected cells was proved to be significantly decreased (P < 0.01 vs. the control group). By the immunohistochemistry method (SABC) to study TGF beta1, positive particles were detected in the experimental group, but there were no positive particles in the control and the blank groups. There was a significant difference between the two groups of the experiment and the control group based on the analysis of the t-test (P < 0.01). By the immunohistochemistry method (SABC) to study collagen II, there were more positive particles in the transfected cells in the experimental group than in the control and the blank groups, and there was a significant difference between the experimental group and the two other groups based on the t-test (P < 0.01).
Transfection of the rabbit MSCs with the reconstructed PGL3-TGF-beta1 gene by the Liposomes Method is successful. There may be some damage to the cells when transfection is performed. The transfected BMS cells with PGL3-TGF-beta1 gene can express and excrete TGF-beta1 when cultured in vitro. The transfected MSCs that secret TGF-beta1 can be self-induced into the chondrocytes after being infected for 7 days when cultured in vitro.
探索用重组PGL3 - 转化生长因子β1（TGF - β1）基因转染骨髓基质干细胞（MSCs）的实验方法，并评估MSCs在体外自我诱导分化为软骨细胞的可行性，为进一步开展“基因增强组织工程”研究提供科学实验依据。
采用脂质体法将重组PGL3 - TGF - β1基因转染兔MSCs，观察细胞生长情况并绘制生长曲线。用MTT法评估实验组转染细胞的活性，与对照组比较差异有统计学意义。转染PGL3 - TGF - β1基因后，在细胞培养2天和7天时，采用免疫组织化学方法（SABC）检测TGF - β1和Ⅱ型胶原抗原。用图像分析仪器分析免疫组织化学切片图像，并用SPSS 11.0软件进行统计学分析，与对照组和空白组比较。
采用脂质体法将重组PGL3 - TGF - β1基因体外转染培养的兔MSCs成功，检测到荧光素酶活性，与对照组比较差异有统计学意义（P < 0.01）。MTT检测显示转染细胞活性明显降低（与对照组比较，P < 0.01）。免疫组织化学方法（SABC）检测TGF - β1，实验组有阳性颗粒，对照组和空白组无阳性颗粒，两组比较差异有统计学意义（t检验，P < 0.01）。免疫组织化学方法（SABC）检测Ⅱ型胶原，实验组转染细胞阳性颗粒多于对照组和空白组，实验组与其他两组比较差异有统计学意义（t检验，P < 0.01）。
采用脂质体法将重组PGL3 - TGF - β1基因转染兔MSCs成功，转染时细胞可能有一定损伤。转染PGL3 - TGF - β1基因的骨髓基质细胞在体外培养时可表达并分泌TGF - β1。分泌TGF - β1的转染MSCs在体外培养7天后可自我诱导分化为软骨细胞。
