共培养大鼠睾丸体细胞组织工程技术构建雄激素分泌组织

Androgen-secreting tissue construction with co-cultured rat testes somatic cells by tissue engineering technique

目的探讨应用组织工程技术体内外构建具有一定形态和睾酮分泌功能组织的可行性。方法雄性 Wister 大鼠,无菌切取双侧睾丸,制备去势及移植鼠动物模型。睾丸去包膜,剪碎,胶原酶消化,经差速贴壁或直接混合共培养方法,分别获取睾丸间质细胞(Leydig 细胞)和共培养的睾丸多种体细胞,两类细胞分别与可降解生物材料聚羟基乙酸(PGA)纤维复合并进行体外培养,光、电镜观察体外组织形成过程,定期检测培养液内睾酮分泌水平。取体外培养7 d 细胞-材料复合物,分别移植于去势鼠睾丸鞘膜腔或大网膜内(n=6),不同时间点取材,通过大体观察、组织学染色和免疫细胞化学方法,检测移植鼠体内雄激素分泌组织的形成过程及血睾酮水平。结果两类细胞均与PGA 具有良好的亲和性,体外可形成具有一定睾酮分泌功能的细胞一材料复合物,体内移植2个月后,两类细胞-材料复合物在大网膜和睾丸鞘膜内均可形成具有一定形态的雄激素分泌组织,再生组织具有良好的血管化。血睾酮检测及统计分析结果表明,移植6周后,单纯 Leydig 细胞组血睾酮水平为(0.60±0.04)μg/L,共培养细胞组血睾酮水平为(0.85±0.03)μg/L,均明显高于单纯去势鼠血睾酮水平(0.56±0.05)μg/L(P<0.01),两组移植鼠相比,共培养细胞移植组血睾酮水平明显高于单纯Leydig 细胞移植组(P<0.01)。结论以共培养睾丸体细胞作为种子细胞在体内外构建雄激素分泌组织具有可行性,共培养睾丸内体细胞是雄激素分泌组织构建的良好种子细胞。

Objective To explore the feasibility of constructing androgen-secreting tissue of a certain size and shape using co-cultured somatic cells of rat testis. Methods Thirty male Wister rats were castrated, model and implanted rat model were prepared by resecting bilateral testes. The suspension of mixed testes cells was cultured to obtain various somatic cells of testes and Leydig cells were collected by differential anchorage-dependent method. These two kinds of cells were seeded onto biodegradable scaffolds of polyglycolic acid （PGA） fibers and cultured in vitro. The tissue formation of cell-scaffold constructs was observed by optical microscope and electronic microscope and the level of testosterone in the supernatant was detected regularly. After 7-day culture in vitro, the 2 kinds of cell-scaffold constructs, scaffold with purified Leydig cells or co-cultured testis somatic cells （ seed cells）, were implanted into the gastrocolic omentum or cavity of tunics vagiuslis of the castrated rats. The implants were harvested 4, 6, 9, 12, and 24 weeks later to evaluate the tissue formation of cell-scaffold constructs in vivo. The serum testosterone level of the implanted rats was assayed to evaluate the testosterone secreting function of the regenerative tissue. Results Both the co-cultured testis somatic cells and Leydig cells had fine compatibility with the PGA fibers and adhered to the scaffolds very welL Testosterone was detected at a certain degree in the superuatant of cell-scaffold constructs, indicating the testosterone secreting function of the constructs. Two months afterthe implantation both kinds of cell-scaffold constructs formed testosterone secreting tissue in both gastrocolic omentum and cavity of tunica vaginalis of the implanted rats. The regenerative tissues were vascularized very well with a certain size and shape. Six weeks after implantation the serum testosterone level of the Leydig cell group was 0. 60 ng/ml ± 0.04 ng/ml, and that of the co-culture group was 0. 84 ng/ml ± 0. 03 ng/ml, both significantly higher than that of the control castrated rats （0. 56 ng/ml ±0. 05 ng/ml, both P 〈0.01 ）, and the serum testosterone level of the co-cultured testes somatic cell implantation group was significantly higher than that of the Leydig cell implantation group too （ P 〈 0. 01 ）. Conclusion It is completely feasible to construct androgen-secreting tissue in vitro and in vivo using tissue engineering technique. Co-cultured testis somatic cells may serve as the better seed cells for androgen-secreting tissue engineering than purified Leydig cells in terms of the quantity and function of cells.