人自体血清培养的鼻中隔软骨细胞

Nasoseptal chondrocytes cultured by human autologous serum

目的:比较人自体血清和胎牛血清对人鼻中隔软骨细胞增殖和分化的影响。方法:实验于2004-08/2005-03在解放军第一军医大学珠江医院全军儿科中心实验室完成。①全血标本取自健康鼻中隔偏曲手术患者,男2例(分别为27岁和32岁),女1例(35岁),经患者知情同意。②自体血清的制备:抽取患者全血40mL,室温静置1h后,离心(1000r/min,30min)后取上清约20mL。③取手术患者的鼻中隔软骨分离软骨细胞进行原代培养,设人自体血清组和胎牛血清组,分别加含体积分数为0.1的自体血清和胎牛血清的1640培养基,并进行传代培养。④取第3代软骨细胞接种于培养板上培养,设人自体血清组、胎牛血清组和对照组(无血清培养),分别于培养后的第1,2,3,4,5,6天行四甲基偶氮唑盐检测。⑤取第2,3,4,5,6代软骨细胞接种于养板上培养,设人自体血清组、胎牛血清组和对照组,行软骨基质蛋白多糖含量(35S-Na2SO4掺入量)测定。⑥取第2代软骨细胞,分别将人自体血清组和胎牛血清组的细胞接种于两个25mL的培养瓶中培养至细胞传代老化,观察细胞表型。结果:①在体积分数为0.1的血清浓度下,人自体血清和胎牛血清对人鼻中隔软骨细胞增殖作用差异不明显(P>0.05)。②人鼻中隔软骨细胞蛋白多糖合成量的比较显示两种血清对第2,3,4,5代软骨细胞软骨基质蛋白多糖的合成作用差异不明显(P>0.05),但在第6代,人自体血清组软骨细胞蛋白多糖合成量明显高于胎牛血清组(P<0.01)。③胎牛血清人鼻中隔软骨细胞到第6代几乎所有细胞变为梭形,人自体血清培养的软骨细胞到第7代以后才绝大部分变为梭形细胞,初步证实了人自体血清在维持鼻中隔软骨细胞表型方面也有一定的作用。结论:人自体血清能够有效地促进软骨细胞增殖和蛋白多糖合成同时维持细胞表型。

AIM： To compare the effects of human autologous serum （AS） and fetal bovine serum （FSC） on the proliferation and differentiation of human nasoseptal chondrocytes. METHODS： The experiment was conducted in the Central Laboratory of Pediatrics of the Whole Army, Zhujiang Hospital Affiliated to First Military Medical University of Chinese PLA between August 2004 and March 2005. ①Sample of whole blood （WB） was obtained from healthy patients with deviated nasal septum, including 2 males （who were 27 and 32 years old respectively） and one female （35 years old）. All patients knew and agreed with the items. ②Preparation of AS： 40 mL of WB obtained from patients were centrifuged （at 1 000 r/minutes for 30 minutes） at one hour after standing at room temperature, from which 20 mL of supernatant were obtained. ③The chondrocytes were isolated from the nasoseptal cartilage of patients for primary culture, which were divided into human AS group and FCS group. 1640 nutrient medium of 10% AS and FCS were added into the two groups respectively for serial subcuhivation, ④The chondrocytes of the third generation were inoculated to the cultural board and cultured respectively as the human AS group, FSC group and control group （non-serum culture）, and detected with MTT respectively at 1, 2, 3, 4, 5, 6 days after culture.⑤The chondrocytes of the 2^nd, 3^rd, 4^th, 5^th and 6^th generations were incubated into the cultural board and cultured as AS group, FCS group and control group. The content of proteoglycan in cartilage matrix （^35SNa2SO4 incorporation） were determined. ⑥ The chondrocytes of the 2nd generation from human AS group and FCS group were inoculated into two 25-mL cultural bottles respectively until cells aging, and the phenotypes of cells were observed. RESULTS： ① Under 10% serum, the effects of human AS and FCS on the proliferation of human nasoseptal chondrocytes were not obvious （P 〉 0.05）.②Comparison in synthesis of proteoglyean in human nasoseptal ehondrocytes showed that the differences in effects on the synthesis of chondrocytes proteoglycan of the 2^nd, 3^rd, 4^th and 5^th generations were not significant between AS and FCS （P 〉 0.05）, whereas the ehondrocytes proteoglycan synthesis of the 6th generation was remarkably higher in the human AS group than that in the FCS group （P 〈 0.01）.⑥ Almost all human nasoseptal chondrocytes in the FCS group were in shuttle at the 6^th generation, while the nasoseptal chondrocytes in the human AS group were not in shuttle until the 7^th generation, which proved that there were certain effect of human AS in maintaining the phenotype of nasoseptal chondrocytes. CONCLUSION： Human AS can effectively promote the proliferation of chondrocytes and the synthesis of proteoglycan as well as maintain the phenotype of cells.