TGF-SAP对平滑肌细胞增殖和内膜增生的抑制作用

Receptor-targeted Cytotoxin-TGFα-SAP Inhibits Smooth Muscle Cell Proliferation and Neointimal Hyperplasia

目的 探讨生物导向药物TGFα SAP对平滑肌细胞增殖及动脉损伤后内膜增生的特异性抑制作用。方法 用SPDP化学联结的方法合成TGFα SAP ,采用细胞计数方法观察TGFα SAP对培养中的增殖平滑肌细胞的细胞毒性作用 ,并以3H leucine渗入法进一步了解TGFα SAP对平滑肌细胞的蛋白质合成的影响 ;在体实验中将Sprague Dawle大鼠随机分为治疗组和对照组 ,均行右颈总动脉球囊内膜剥脱术 ,治疗组术后局部注射TGFα SAP ,对照组予以生理盐水 ;于不同时间点处死动物取动脉段行光镜观察及计算机图像分析。结果 从体外实验可见TGFα SAP能明显抑制SMCs的生长增殖及蛋白质合成 ;在体实验中图像分析结果显示 :动脉损伤后TGFα SAP治疗组在第 3 ,9和 2 8天内膜 /内膜加中膜面积显著小于对照组 (P <0 .0 5 )。结论 TGFα SAP与Saporin相比对增殖平滑肌细胞具有明显增强的细胞毒性 。

Objective To testify the special citotoxicity of TGFα SAP on proliferating vascular smooth muscle cells in vitro and effective inhibition of TGFα SAP on neointimal hyperplasia after arterial injury in vivo. Methods Conjugation of saporin to TGFα was accomplished after derivatization of saporin and TGFα with N succinimidyl 3 (2 pyridyldithio) proprionate and final purification of the conjugate was achieved within Eppendorf Centrifugal Filter Tubes. Biological assay of cytotoxicity assays was measured by cell count. The studies of influence of TGFα SAP on values of leucine incorporation into SMCs were measured by 3H leucine uptake. In vivo, forty eight rats were randomly divided into treatment group and control group. Rats in the treatment group were treated by local injection of TGFα SAP after arterial injury and those in the control group were treated by saline. Optical microscope and computer image analysis were used to study rat arterial segment 1,3,9 and 28 days after treat.ment. Results In vitro, biological assay of cytotoxicity assays testified TGFα SAP conjugate could inhibit specially proliferation of SMCs in culture. The cell number of the group treated by TGFα SAP (10 -7 mol/L) reduced to 45.1% compared with the control group. But Saporin (10 -3 mol/L) did not affect cellular proliferation. The values of leucine of TGFα SAP group (10 -9 M) in comparison significantly decreased to 47.3% of the control group ( P <0.05), suggesting that cellular protein synthesis obviously decreased as TGFα SAP was added. In vivo, significant inhibitions of intimal proliferation and less stenosis of arterial cavity of the treatment group were observed by optical microscope. Computer image analysis showed that the ratio of neointimal area to intima area and media area of the treatment group 1,3,9 and 28 days after treatment was significant smaller than that of control ( P <0.05) .Conclusion The results indicated that TGFα SAP possesses the more effective cytotocxicity on proliferating SMCs than saporin. TGFα SAP can effectively inhibit SMCs proliferation and neointimal hyperplasia after arterial injury in vivo.