本文關(guān)鍵詞：低氧對(duì)大鼠髁突軟骨細(xì)胞HIF-1α，VEGF，aggrecanase-1和TIMP-3的表達(dá)的影響　出處：《廣西醫(yī)科大學(xué)》2011年碩士論文　論文類型：學(xué)位論文

  更多相關(guān)文章： 低氧誘導(dǎo)因子-1α 血管內(nèi)皮生長(zhǎng)因子 聚集蛋白聚糖酶-1 金屬蛋白酶抑制因子-3 低氧 髁突軟骨 二氯化鈷

【摘要】：目的分離、培養(yǎng)及鑒定原代大鼠髁突軟骨細(xì)胞,建立二氯化鈷(CoCl2)化學(xué)低氧的細(xì)胞培養(yǎng)模型,研究體外常氧與低氧條件下培養(yǎng)髁突軟骨細(xì)胞低氧誘導(dǎo)因子-1α(hypoxia inducible factor-1α, HIF-1α)、血管內(nèi)皮生長(zhǎng)因子(vascular endothelial growth factor, VEGF)、聚集蛋白聚糖酶-1(aggrecanase-1)和金屬蛋白酶抑制劑-3(tissue inhibitor of metalloproteinase-3, TIMP-3)的表達(dá)變化,探討其在髁突軟骨發(fā)育中的意義。 方法(1)采用機(jī)械分離、胰蛋白酶和Ⅱ型膠原酶聯(lián)合消化,從三周齡SD大鼠髁突分離出軟骨細(xì)胞,并進(jìn)行原代和傳代培養(yǎng);倒置顯微鏡觀察細(xì)胞的形態(tài)及其生長(zhǎng)情況,MTT比色法繪制髁突軟骨細(xì)胞的生長(zhǎng)曲線,利用Ⅱ型膠原免疫組織化學(xué)染色和甲苯胺藍(lán)染色進(jìn)行鑒定。 (2)運(yùn)用含不同濃度CoCl2的培養(yǎng)基75μmol/L(A組)、125μmol/L(B組)、175μmol/L(C組)以及0μmol/L(常氧組)分別進(jìn)行細(xì)胞培養(yǎng),48h后測(cè)定并比較各組的乳酸脫氫酶(LDH)釋放率,建立CoCl2化學(xué)低氧的細(xì)胞培養(yǎng)模型。 (3)采用逆轉(zhuǎn)錄-聚合酶鏈反應(yīng)(RT-PCR)檢測(cè)常氧狀態(tài)下與低氧后12h、24h、48h各個(gè)時(shí)間段原代大鼠髁突軟骨細(xì)胞HIF-1α、VEGF、aggrecanase-1和TIMP-3的mRNA的表達(dá)差異。 結(jié)果(1)此方法分離每只大鼠髁狀突關(guān)節(jié)可以獲得1×10~4～4×10~4個(gè)軟骨細(xì)胞,臺(tái)盼藍(lán)染色獲得的原代細(xì)胞存活率達(dá)到95%。甲苯胺藍(lán)染色可見髁突軟骨細(xì)胞核被染成深藍(lán)色,胞漿及細(xì)胞外基質(zhì)被異染成紫紅色。Ⅱ型膠原免疫組化染色可見髁突軟骨胞質(zhì)出現(xiàn)陽(yáng)性染色。 (2)在48h時(shí)間點(diǎn)上,隨著CoCl2的濃度增高,LDH釋放率增高,C組的LDH釋放率明顯高于常氧組(P0.01),A組、B組的LDH釋放率分別與常氧組比較差異無統(tǒng)計(jì)學(xué)意義(P0.05)。 (3)半定量RT-PCR結(jié)果顯示,在常氧條件下培養(yǎng)的髁突軟骨細(xì)胞HIF-1α、VEGF、aggrecanase-1和TIMP-3均有表達(dá);與常氧條件下相比,髁突軟骨細(xì)胞在低氧條件下培養(yǎng),在低氧后12h、24h、48h時(shí)間點(diǎn)HIF-1α(P0.05)、VEGF(P0.01)的mRNA表達(dá)明顯升高;并且VEGF(P0.01)、HIF-1α(P0.05)在12h與24h、12h與48h、24h與48h比較表達(dá)均有統(tǒng)計(jì)學(xué)差異;HIF-1α表達(dá)逐次升高,而VEGF在12 h達(dá)到最高點(diǎn)后逐次下降,但是均高于常氧狀態(tài)下。Aggrecanase-1和TIMP-3的表達(dá)在各個(gè)時(shí)間點(diǎn)之間與常氧狀態(tài)下沒有統(tǒng)計(jì)學(xué)差異。 結(jié)論(1)本研究培養(yǎng)髁突軟骨細(xì)胞獲取大量存活率高的軟骨細(xì)胞。 (2)LDH釋放率結(jié)果提示175μmol/L處理軟骨細(xì)胞后對(duì)細(xì)胞產(chǎn)生嚴(yán)重的損傷,不適用于建立低氧模型,而由于大多數(shù)文獻(xiàn)報(bào)道75μmol/L CoCl2產(chǎn)生的低氧效果不如125μmol/L CoCl_2,所以最終確定125μmol/L作為建立低氧模型的濃度。利用CoCl2建立的髁突軟骨細(xì)胞化學(xué)低氧模型,簡(jiǎn)單、穩(wěn)定并且易于操作。 (3)低氧極早期可上調(diào)髁突軟骨細(xì)胞HIF-1α、VEGF mRNA表達(dá)。但是aggrecanase-1和TIMP-3的表達(dá)是多因素調(diào)節(jié)的結(jié)果,體外的缺氧模型尚不能完全模擬體內(nèi)精細(xì)復(fù)雜的調(diào)控機(jī)制,低氧極早期對(duì)髁突軟骨aggrecanase-1和TIMP-3 mRNA的表達(dá)無明顯影響。HIF-1α、VEGF與髁突軟骨細(xì)胞適應(yīng)低氧環(huán)境密切相關(guān),可能在髁突軟骨血管形成以及生長(zhǎng)發(fā)育發(fā)揮重要作用。但隨時(shí)間延長(zhǎng),VEGF的表達(dá)并未隨HIF-1α逐漸升高。
[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.

【學(xué)位授予單位】：廣西醫(yī)科大學(xué)
【學(xué)位級(jí)別】：碩士
【學(xué)位授予年份】：2011
【分類號(hào)】：R363

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