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山西醫(yī)科大第二醫(yī)院丨DAla2GIP抑制鈣吸收拮抗軟骨細(xì)胞凋亡
NMT是基因功能的活體檢測(cè)技術(shù)，已被103位諾貝爾獎(jiǎng)得主所在單位，及北大、清華、中科院使用。
期刊：Bone
主題：DAla2GIP抑制鈣吸收拮抗軟骨細(xì)胞凋亡
標(biāo)題：DAla2GIP antagonizes H2O2-induced chondrocyte apoptosis and inflammatory factor secretion
影響因子：4.360
檢測(cè)指標(biāo)：Ca2+流速
檢測(cè)樣品：7日齡小鼠，1.0ml乙麻醉后處死，第三代胸部軟骨組織細(xì)胞
Ca2+流實(shí)驗(yàn)處理方法：
Control，300μM H2O2 ，100pM DAla2GIP 和 300μM H2O2+ 100pM DAla2GIP
Ca2+流實(shí)驗(yàn)測(cè)試液成份：無
作者：山西醫(yī)科大學(xué)第二醫(yī)院衛(wèi)小春、王宇澤
英文摘要
To investigate the protective effects of DAla2GIP against the apoptosis and inflammatory factor secretion in H2O2-induced chondrocyte, and explore the possible mechanisms of DAla2GIP underlying its protection.
The chondrocytes were divided into the following four groups: Control, 300?μM H2O2, 100?pM DAla2GIP and 300?μM H2O2?+?100?pM DAla2GIP. The apoptosis of chondrocyte was measured by using mitochondrial membrane potential assay kit (JC-1) and TUNEL assay, the inflammatory factor secretion were assessed by ELISA assay, and the cellular and molecular mechanisms of DAla2GIP protection were investigated by using Real time-PCR, flow cytometry, Non- invasive calcium detection and western blotting techniques.
(1) DAPla2GIP prevents apoptosis of chondrocyte induced by H2O2. (2) DAla2GIP alleviated the inflammation of chondrocyte induced by H2O2. (3) DAla2GIP prevents chondrocyte apoptosis by inhibiting calcium influx of chondrocyte and regulating expression of Bcl-2 and Caspase-3induced by H2O2. (4) DAla2GIP inhibited the H2O2 mediated inflammation by up- regulating the expressions of Sox9 and Col2a1 and inhibiting PI3K/Akt/NF-κB pathway.
Our experimental results revealed that DAla2GIP prevents chondrocyte apoptosis by inhibiting calcium influx of chondrocyte and induced regulating expression of Bcl-2 and Casp ase-3by H2O2. Further, molecular biology experiments confirmed that DAla2GIP inhibited the H2O2 mediated inflammation vis up-regulating the expressions of Sox9 and Col2a1 and inhibiting PI3K/Akt/NF-κB pathway. The results demonstrate that DAla2GIP has protective properties in H2O2-induced chondrocyte injury, this finding shows that novel GIP analogues have the potential as a novel therapeutic for osteoarthritis patients.
中文摘要（谷歌機(jī)翻）
研究DAla2GIP對(duì)H2O2誘導(dǎo)的軟骨細(xì)胞凋亡和炎性因子分泌的保護(hù)作用，并探討DAla2GIP保護(hù)其的可能機(jī)制。
軟骨細(xì)胞分為以下四組：對(duì)照組，300μmH2O2、100μmM DAla2GIP和300μmH2O2n +100μpMDAla2GIP。用線粒體膜電位分析試劑盒（JC-1）和TUNEL法檢測(cè)軟骨細(xì)胞的凋亡，用ELISA法評(píng)估炎性因子的分泌，并通過實(shí)時(shí)PCR研究DAla2GIP保護(hù)的細(xì)胞和分子機(jī)制，流式細(xì)胞儀，無創(chuàng)鈣離子檢測(cè)和蛋白質(zhì)印跡技術(shù)。
（1）DAPla2GIP可防止H2O2誘導(dǎo)的軟骨細(xì)胞凋亡。（2）DAla2GIP減輕了H2O2誘導(dǎo)的軟骨細(xì)胞炎癥。 （3）DAla2GIP通過抑制軟骨細(xì)胞的鈣內(nèi)流并調(diào)節(jié)過氧化氫誘導(dǎo)的Bcl-2和Caspase-3的表達(dá)來防止軟骨細(xì)胞凋亡。（4）DAla2GIP通過上調(diào)Sox9和Col2a1的表達(dá)并抑制PI3K / Akt /NF-κB通路來抑制H2O2介導(dǎo)的炎癥。
我們的實(shí)驗(yàn)結(jié)果表明，DAla2GIP通過抑制軟骨細(xì)胞的鈣內(nèi)流并誘導(dǎo)H2O2調(diào)節(jié)Bcl-2和Casp ase-3的表達(dá)來防止軟骨細(xì)胞凋亡。此外，分子生物學(xué)實(shí)驗(yàn)證實(shí)，DAla2GIP通過上調(diào)Sox9和Col2a1的表達(dá)并抑制PI3K / Akt /NF-κB通路來抑制H2O2介導(dǎo)的炎癥。結(jié)果表明，DAla2GIP在H2O2誘導(dǎo)的軟骨細(xì)胞損傷中具有保護(hù)性，這一發(fā)現(xiàn)表明，新型GIP類似物具有作為骨關(guān)節(jié)炎患者的新型治療劑的潛力。
結(jié)果表明：100 pM DAla2-GIP組和Control組的Ca2+吸收速率并無顯著差異，平均值分別是1.94 pmol·cm-2·s-1和1.42 pmol·cm-2·s-1；經(jīng)300 μM H2O2處理后，Ca2+吸收速率明顯增加，平均值為139.61 pmol·cm-2·s-1；經(jīng)300 μM H2O2和100 pM DAla2-GIP共同處理后，Ca2+吸收速率明顯低于300 μM H2O2處理組，平均值為26.68 pmol·cm-2·s-1，說明DAla2-GIP抑制了H2O2誘導(dǎo)的Ca2+吸收。而Ca2+吸收是軟骨細(xì)胞凋亡早期的標(biāo)志。