【摘要】：目的：探討成骨細胞和脂肪細胞兩者間橫向分化的能力和方法,為進一步行縫隙連接通訊對其調(diào)控的實驗做準備。 方法：選取3月齡新西蘭大白兔腹股溝處脂肪組織進行分離培養(yǎng)脂肪細胞,天花板貼壁法對脂肪細胞進行細胞去分化培養(yǎng),利用第3代去分化脂肪細胞進行成骨誘導(dǎo),并設(shè)立對照組進行對比。成骨分化指標的檢測：對培養(yǎng)3周后的兩組細胞進行Ⅰ型膠原免疫組化染色；使用BCIP/NBT ALP顯色試劑盒對兩組細胞進行ALP染色；使用ALP活性檢測試劑盒分別檢測各組第7天、第14天、第21天細胞的ALP活性；使用茜素紅對連續(xù)培養(yǎng)3周的兩組細胞進行鈣結(jié)節(jié)染色。取出生7天內(nèi)的乳兔顱骨進行成骨細胞的獲得及培養(yǎng),利用第3代成骨細胞細胞進行成脂誘導(dǎo),并設(shè)立對照組對比。脂肪細胞分化指標的檢測：兩組細胞培養(yǎng)3周后行油紅0染色；RT-PCR檢測兩組細胞PPARγmRNA的表達。 結(jié)果：1、提取的成熟脂肪細胞經(jīng)天花板貼壁法培養(yǎng)后形態(tài)為長梭形成纖維細胞狀。2、成骨分化檢測：Ⅰ型膠原免疫組化染色顯示實驗組細胞內(nèi)表達出Ⅰ型膠原,與對照組比較有統(tǒng)計學意義(p0.05)；ALP活性檢測試劑盒檢測各組細胞不同時段的ALP活性發(fā)現(xiàn)實驗組較對照組高(p0.05)；且隨著培養(yǎng)時間的增長實驗組ALP活性逐漸增強(組內(nèi)p0.05),與對照組比較均有統(tǒng)計學意義(p0.05)；經(jīng)BCIP/NBT ALP染色試劑盒檢測,實驗組細胞染色有棕黑色細微顆粒,多數(shù)分布在細胞膜上以及周圍,對照組細胞染色無棕黑色顆粒出現(xiàn);茜素紅對連續(xù)培養(yǎng)3周的兩組細胞進行鈣結(jié)節(jié)染色發(fā)現(xiàn)實驗組有較多橘紅色結(jié)節(jié),而對照組無明顯發(fā)現(xiàn)。3、對提取的乳兔顱骨原代細胞茜素紅染色后證實提取的細胞為成骨細胞。4、成脂檢測指標：油紅0染色顯示實驗組細胞內(nèi)出現(xiàn)大量紅染顆粒,對照組細胞未被紅染; RT-PCR檢測兩組細胞PPARγmRNA的表達,實驗組可見有PPARγmRNA表達,而對照組無PPARγmRNA的表達。 結(jié)論：成熟脂肪細胞可以通過體外天花板培養(yǎng)實現(xiàn)去分化,去分化的脂肪細胞在成骨誘導(dǎo)環(huán)境下可以向成骨細胞分化；成骨細胞在成脂誘導(dǎo)環(huán)境下可以分化為脂肪細胞；成骨細胞和脂肪細胞在一定條件下可以相互轉(zhuǎn)化。
[Abstract]:Aim: to investigate the ability and method of lateral differentiation between osteoblasts and adipocytes in order to prepare for the regulation of gap junction communication. Methods: adipocytes were isolated from the inguinal adipocytes of New Zealand white rabbits at 3 months old. Adipocytes were dedifferentiated and induced by the third generation of dedifferentiated adipocytes. A control group was set up for comparison. Detection of osteogenic differentiation: type I collagen immunohistochemical staining was performed on two groups of cells after 3 weeks of culture; ALP staining was performed on both groups using BCIP/NBT ALP reagent kit; ALP activity assay kit was used to detect 7 days in each group, respectively. The activity of ALP was observed on day 14 and day 21, and calcium nodules were stained by alizarin red in two groups of cells cultured for 3 weeks. Osteoblasts were obtained and cultured from the calvaria of newborn rabbits within 7 days. The third generation of osteoblasts were used to induce adipogenesis and the control group was set up. Detection of adipocyte differentiation index: after 3 weeks of culture, the expression of PPAR 緯 mRNA in the two groups was detected by oil red 0 staining reverse transcription-polymerase chain reaction (RT-PCR). Results: the mature adipocytes cultured by ceiling adherent method showed long fusiform fibroblasts. The osteogenic differentiation was detected. The expression of type 鈪，

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