低氧对大鼠髁突软骨细胞HIF-1α，VEGF，aggrecanase-1和TIMP-3的表达的影响

发布时间：2018-01-15 01:04

本文关键词：低氧对大鼠髁突软骨细胞HIF-1α，VEGF，aggrecanase-1和TIMP-3的表达的影响　出处：《广西医科大学》2011年硕士论文　论文类型：学位论文

【摘要】：目的分离、培养及鉴定原代大鼠髁突软骨细胞,建立二氯化钴(CoCl2)化学低氧的细胞培养模型,研究体外常氧与低氧条件下培养髁突软骨细胞低氧诱导因子-1α(hypoxia inducible factor-1α, HIF-1α)、血管内皮生长因子(vascular endothelial growth factor, VEGF)、聚集蛋白聚糖酶-1(aggrecanase-1)和金属蛋白酶抑制剂-3(tissue inhibitor of metalloproteinase-3, TIMP-3)的表达变化,探讨其在髁突软骨发育中的意义。方法(1)采用机械分离、胰蛋白酶和Ⅱ型胶原酶联合消化,从三周龄SD大鼠髁突分离出软骨细胞,并进行原代和传代培养;倒置显微镜观察细胞的形态及其生长情况,MTT比色法绘制髁突软骨细胞的生长曲线,利用Ⅱ型胶原免疫组织化学染色和甲苯胺蓝染色进行鉴定。 (2)运用含不同浓度CoCl2的培养基75μmol/L(A组)、125μmol/L(B组)、175μmol/L(C组)以及0μmol/L(常氧组)分别进行细胞培养,48h后测定并比较各组的乳酸脱氢酶(LDH)释放率,建立CoCl2化学低氧的细胞培养模型。 (3)采用逆转录-聚合酶链反应(RT-PCR)检测常氧状态下与低氧后12h、24h、48h各个时间段原代大鼠髁突软骨细胞HIF-1α、VEGF、aggrecanase-1和TIMP-3的mRNA的表达差异。结果(1)此方法分离每只大鼠髁状突关节可以获得1×10~4～4×10~4个软骨细胞,台盼蓝染色获得的原代细胞存活率达到95%。甲苯胺蓝染色可见髁突软骨细胞核被染成深蓝色,胞浆及细胞外基质被异染成紫红色。Ⅱ型胶原免疫组化染色可见髁突软骨胞质出现阳性染色。 (2)在48h时间点上,随着CoCl2的浓度增高,LDH释放率增高,C组的LDH释放率明显高于常氧组(P0.01),A组、B组的LDH释放率分别与常氧组比较差异无统计学意义(P0.05)。 (3)半定量RT-PCR结果显示,在常氧条件下培养的髁突软骨细胞HIF-1α、VEGF、aggrecanase-1和TIMP-3均有表达;与常氧条件下相比,髁突软骨细胞在低氧条件下培养,在低氧后12h、24h、48h时间点HIF-1α(P0.05)、VEGF(P0.01)的mRNA表达明显升高;并且VEGF(P0.01)、HIF-1α(P0.05)在12h与24h、12h与48h、24h与48h比较表达均有统计学差异;HIF-1α表达逐次升高,而VEGF在12 h达到最高点后逐次下降,但是均高于常氧状态下。Aggrecanase-1和TIMP-3的表达在各个时间点之间与常氧状态下没有统计学差异。结论(1)本研究培养髁突软骨细胞获取大量存活率高的软骨细胞。 (2)LDH释放率结果提示175μmol/L处理软骨细胞后对细胞产生严重的损伤,不适用于建立低氧模型,而由于大多数文献报道75μmol/L CoCl2产生的低氧效果不如125μmol/L CoCl_2,所以最终确定125μmol/L作为建立低氧模型的浓度。利用CoCl2建立的髁突软骨细胞化学低氧模型,简单、稳定并且易于操作。 (3)低氧极早期可上调髁突软骨细胞HIF-1α、VEGF mRNA表达。但是aggrecanase-1和TIMP-3的表达是多因素调节的结果,体外的缺氧模型尚不能完全模拟体内精细复杂的调控机制,低氧极早期对髁突软骨aggrecanase-1和TIMP-3 mRNA的表达无明显影响。HIF-1α、VEGF与髁突软骨细胞适应低氧环境密切相关,可能在髁突软骨血管形成以及生长发育发挥重要作用。但随时间延长,VEGF的表达并未随HIF-1α逐渐升高。
[Abstract]:Objective to isolate, culture and identification of primary rat condylar cartilage cells, the establishment of two cobalt chloride (CoCl2) chemical hypoxia cell culture model, condylar cartilage cell hypoxia inducible factor -1 alpha cultured in vitro under normoxia and hypoxia (hypoxia inducible factor-1 HIF-1 alpha, alpha), vascular endothelial growth factor (vascular endothelial growth factor, VEGF), aggrecanase -1 (aggrecanase-1) and metalloproteinase inhibitor -3 (tissue inhibitor of metalloproteinase-3, TIMP-3) expression on the development of the condylar cartilage.
Methods (1) by mechanical separation, trypsin and type II collagenase digestion, chondrocytes were isolated from three week old SD rat condyle, and cultured; morphology and growth of cells were observed under inverted microscope. The growth curve, MTT assay of condylar cartilage cells, immunohistochemical identification the use of type II collagen immunohistochemical staining and toluidine blue.
(2) cell culture was carried out with different medium CoCl2, 75 mol/L (group A), 125 mol/L (B group), 175 mol/L (C group) and 0 mol/L mol/L (normoxic group). After 48h, the lactate dehydrogenase (LDH) release rate of each group was detected and compared, and the cell culture model of CoCl2 chemical hypoxia was established.
(3) reverse transcription polymerase chain reaction (RT-PCR) was used to detect the expression differences of mRNA, HIF-1, VEGF, aggrecanase-1 and TIMP-3 in primary rat condyle chondrocytes in normoxic state and 12h, 24h and 48h after hypoxia.
Results (1) the separation method of each rat condylar joint can be obtained in 1 ~ 4 * 10~4 * 10~4 cartilage cells, trypan blue staining in the primary cell survival rate of 95%. toluidine blue staining showed that the condylar cartilage cell nuclei were stained dark blue, cytoplasm and extracellular matrix by different purple red. Type II collagen immunohistochemical staining of condylar cartilage appeared in cytoplasm staining.
(2) at 48h time point, with the increase of CoCl2 concentration, the LDH release rate increased, and the LDH release rate in C group was significantly higher than that in normoxic group (P0.01). There was no significant difference in LDH release rate between B group and normoxic group (P0.05).
(3) semi quantitative RT-PCR results showed that cultured under normoxia condition of condylar chondrocytes of HIF-1 alpha, VEGF, aggrecanase-1 and TIMP-3 were expressed; compared with normoxia, condylar chondrocytes cultured under hypoxia, hypoxia in 12h, 24h, 48h time point HIF-1 alpha (P0.05). VEGF (P0.01) mRNA expression was significantly increased; and the VEGF (P0.01), alpha HIF-1 (P0.05) in 12h and 24h, 12h and 48h, 24h and 48h expression were statistically significant; the HIF-1 expression of VEGF in 12 successive increases, and H reached the highest point after successive decline, but the expression is higher than normoxia under the condition of.Aggrecanase-1 and TIMP-3 between each time point and under normoxic condition was not statistically significant.
Conclusions (1) in this study, a large number of chondrocytes with high survival rates were obtained by developing condylar chondrocytes.
(2) LDH release rate results suggest that 175 mol/L treatment of cartilage cells cause serious damage to cells, not suitable for the establishment of the model of hypoxia and hypoxia, because most of the reported effects of 75 mol/L CoCl2 than 125 mol/L CoCl_2, so the final 125 mu mol/L as concentration is established. Using the model of hypoxia of condyle cartilage cell chemical hypoxia model, CoCl2 to establish a simple, stable and easy to operate.
(3) early hypoxia can upregulate the condylar cartilage cells of HIF-1 alpha, VEGF expression of mRNA. But the expression of aggrecanase-1 and TIMP-3 are many factors regulating the hypoxic model in vitro cannot fully simulate the regulation mechanism of body complex, very early expression of hypoxia on condylar cartilage aggrecanase-1 and TIMP-3 mRNA had no effect on.HIF-1 alpha, VEGF and condylar cartilage cells adapt to hypoxia is closely related to the environment, may be formed in the condylar cartilage and vascular growth play an important role. But with the extension of time, the expression of VEGF did not change with HIF-1 alpha gradually increased.

【学位授予单位】：广西医科大学
【学位级别】：硕士
【学位授予年份】：2011
【分类号】：R363

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