【摘要】：目的：(1)單層培養(yǎng)條件下，觀察人血小板上清液(human Platelet Supernatant，hPS)對生長板軟骨細(xì)胞增殖、代謝和分化等生物學(xué)活性的影響。(2)在離心成團(tuán)培養(yǎng)系統(tǒng)中，探討hPS對體外形成的生長板軟骨組織增殖、代謝和分化等生物學(xué)活性的影響，評價hPS應(yīng)用于軟骨組織工程的可行性。 方法：(1)原代分離培養(yǎng)兔生長板軟骨細(xì)胞并穩(wěn)定傳代，同時添加不同濃度的hPS，采用WST-8染色法檢測細(xì)胞增殖倍數(shù)：Alcian Blue法測基質(zhì)蛋白多糖含量；羥脯氨酸法測定軟骨細(xì)胞膠原產(chǎn)量；酶動力學(xué)方法測定堿性磷酸酶(alkaline phosphatase，ALP)活性。(2)離心后成團(tuán)培養(yǎng)兔生長板軟骨細(xì)胞，根據(jù)第一部分實(shí)驗(yàn)結(jié)果添加適當(dāng)濃度的hPS。組織切片HE染色，觀察新生軟骨組織形態(tài)和亞細(xì)胞結(jié)構(gòu)；Hoechst33258染色，，檢測DNA含量；羥脯氨酸法測定基質(zhì)膠原含量；Alcian Blue法測定基質(zhì)蛋白多糖含量；組織切片，免疫組化檢測Ⅰ、Ⅱ型膠原表達(dá)；半定量RT-PCR檢測Ⅰ、Ⅱ型膠原mRNA表達(dá)。 結(jié)果：(1)單層培養(yǎng)條件下：體積分?jǐn)?shù)5％hPS實(shí)驗(yàn)組與對照組相比，軟骨細(xì)胞的增殖以及基質(zhì)蛋白多糖的合成顯著增加(P＜0.05)，但形態(tài)上失去其軟骨細(xì)胞分化表型；各濃度組hPS對總膠原的合成與對照組相比沒有差別(P＞0.05)，添加hPS各濃度組與對照組相比ALP的表達(dá)顯著降低。(2)成團(tuán)培養(yǎng)條件下：軟骨細(xì)胞在體外形成了軟骨樣
[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

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