人血小板上清液对家兔软骨细胞的生物学活性研究

发布时间：2018-09-06 18:24

【摘要】：目的：(1)单层培养条件下，观察人血小板上清液(human Platelet Supernatant，hPS)对生长板软骨细胞增殖、代谢和分化等生物学活性的影响。(2)在离心成团培养系统中，探讨hPS对体外形成的生长板软骨组织增殖、代谢和分化等生物学活性的影响，评价hPS应用于软骨组织工程的可行性。 方法：(1)原代分离培养兔生长板软骨细胞并稳定传代，同时添加不同浓度的hPS，采用WST-8染色法检测细胞增殖倍数：Alcian Blue法测基质蛋白多糖含量；羟脯氨酸法测定软骨细胞胶原产量；酶动力学方法测定碱性磷酸酶(alkaline phosphatase，ALP)活性。(2)离心后成团培养兔生长板软骨细胞，根据第一部分实验结果添加适当浓度的hPS。组织切片HE染色，观察新生软骨组织形态和亚细胞结构；Hoechst33258染色，，检测DNA含量；羟脯氨酸法测定基质胶原含量；Alcian Blue法测定基质蛋白多糖含量；组织切片，免疫组化检测Ⅰ、Ⅱ型胶原表达；半定量RT-PCR检测Ⅰ、Ⅱ型胶原mRNA表达。 结果：(1)单层培养条件下：体积分数5％hPS实验组与对照组相比，软骨细胞的增殖以及基质蛋白多糖的合成显著增加(P＜0.05)，但形态上失去其软骨细胞分化表型；各浓度组hPS对总胶原的合成与对照组相比没有差别(P＞0.05)，添加hPS各浓度组与对照组相比ALP的表达显著降低。(2)成团培养条件下：软骨细胞在体外形成了软骨样
[Abstract]:Objective: (1) to observe the effects of human platelet supernatant (human Platelet Supernatant,hPS) on the proliferation, metabolism and differentiation of chondrocytes in monolayer culture. To investigate the effects of hPS on proliferation, metabolism and differentiation of cartilage in vitro, and to evaluate the feasibility of hPS in cartilage tissue engineering. Methods: (1) the rabbit growth plate chondrocytes were isolated and cultured stably. At the same time, the content of matrix proteoglycan was measured by WST-8 staining with different concentrations of hPS,. Collagen production in chondrocytes was measured by hydroxyproline method, alkaline phosphatase (alkaline phosphatase,ALP) activity was measured by enzyme kinetic method. (2) Rabbit growth plate chondrocytes were cultured after centrifugation. According to the results of the first part of the experiment, appropriate concentration of hPS. was added. Tissue sections were stained with HE, and the tissue morphology and subcellular structure of newborn cartilage were observed by Hoechst33258 staining to detect the content of DNA; hydroxyproline method to determine the content of collagen in matrix; Alcian Blue method to determine the content of proteoglycan; The expression of type 鈪

本文编号：2227152