發(fā)布時(shí)間：2018-07-21 11:00

【摘要】：目的 探討?yīng)毣罴纳鷾珜?duì)骨性關(guān)節(jié)炎大鼠關(guān)節(jié)軟骨細(xì)胞增殖與凋亡的影響。 方法 1.健康2月齡清潔級(jí)SD大鼠144只,雌雄各半,適應(yīng)性喂養(yǎng)1周,隨機(jī)分為空白組(36只)和造模組(108只)。造模組在1,4,7天雙膝關(guān)節(jié)腔注射4%木瓜蛋白酶復(fù)制骨性關(guān)節(jié)炎模型。造模成功后,抽簽法隨機(jī)分為模型組(36只)、治療組(36只)和陽(yáng)性組(36只)。空白組和模型組按照10ml·kg-1·d-1的量灌服0.9%生理鹽水；治療組按照9.3g.kg-1·d-1的藥量灌服獨(dú)活寄生湯；陽(yáng)性組按照0.15g-kg-1d-1的藥量灌服奧泰靈膠囊；每2周為1個(gè)療程,中間休息2天,共4個(gè)療程。 2.每2個(gè)療程后,處死一批實(shí)驗(yàn)動(dòng)物,切取脛骨平臺(tái)內(nèi)側(cè)軟骨組織于4%多聚甲醛中固定、石蠟包埋,HE染色觀察軟骨組織形態(tài)變化；免疫組化檢測(cè)Cyclin D1、CDK4、 Rb、p16、Bc1-2、Bax、Cytochrome c、caspase-9及caspase-3蛋白表達(dá)情況；切取脛骨平臺(tái)外側(cè)軟骨組織于電鏡固定液中固定,用于觀察軟骨細(xì)胞超微結(jié)構(gòu)變化；其余軟骨組織迅速置入液氮中保存,用于檢測(cè)Cyclin D1、CDK4、Rb及p16mRNA表達(dá)情況。 3.健康2月齡清潔級(jí)SD大鼠144只,雌雄各半,抽簽法隨機(jī)分為：不用藥血清對(duì)照組(空白血清組,36只)和獨(dú)活寄生湯臨床等效劑量組(含藥血清組,108只)�？瞻籽褰M按10ml·kg-1·d-1的量給予0.9%生理鹽水；含藥血清組按9.3g-kg-1-d-1的量給予獨(dú)活寄生湯。連續(xù)7天灌胃,最后1天連續(xù)2次灌胃,中間間隔2h,采血前禁食12h,分別在末次灌胃1h、2h、3h后,在2%戊巴比妥鈉2ml/kg麻醉下腹主動(dòng)脈采血,制備含藥血清。 4.健康4周齡清潔級(jí)SD大鼠20～30只,取膝關(guān)節(jié)的關(guān)節(jié)軟骨,建立軟骨細(xì)胞體外培養(yǎng)體系。第2代軟骨細(xì)胞以1.0×104/孔接種于96孔板中,在含10%FBS的DMEM培養(yǎng)基中培養(yǎng)24h后,用不含F(xiàn)BS的DMEM培養(yǎng)液培養(yǎng)24h,同步化軟骨細(xì)胞。分別加入含10%、15%、20%、30%四個(gè)濃度的不同采血時(shí)間點(diǎn)的含藥血清和空白血清,培養(yǎng)24h、36h、48h、60h、72h后,MTT法檢測(cè)軟骨細(xì)胞的活性。 5.用10ng/ml IL-1β誘導(dǎo)第3代軟骨細(xì)胞復(fù)制退變軟骨細(xì)胞模型,采用倒置相差顯微鏡觀察細(xì)胞形態(tài)變化和Ⅱ型膠原免疫組化進(jìn)行鑒定。 6.采用(6)中確定的含藥血清最佳干預(yù)條件,體外培養(yǎng)退變軟骨細(xì)胞,分別干預(yù)24h,48h,72h后,收集軟骨細(xì)胞。MTT法檢測(cè)細(xì)胞增殖情況；流式細(xì)胞術(shù)檢測(cè)細(xì)胞周期變化情況；Annexin V-FITC/PI染色流式檢測(cè)細(xì)胞凋亡情況；JC-1染色流式檢測(cè)含藥血清干預(yù)后線粒體膜電位的改變情況; RT-PCR法檢測(cè)Cyclin D1、CDK4、Rb、p16、Bcl-2、 Bax mRNA表達(dá)情況；Western Blot法檢測(cè)Cyclin D1、CDK4、Rb、p16、Bcl-2、Bax蛋白表達(dá)情況；分光光度法檢測(cè)caspase-9和caspase-3活化情況。 結(jié)果 1.膝關(guān)節(jié)X側(cè)位片示,正常組關(guān)節(jié)間隙正常、關(guān)節(jié)面平整、關(guān)節(jié)邊緣可見(jiàn)光滑銳利的線樣致密影,軟骨下骨密度均勻。模型組關(guān)節(jié)間隙明顯變窄、關(guān)節(jié)面不平整,軟骨破壞區(qū)邊緣骨質(zhì)增生硬化,關(guān)節(jié)邊緣骨贅形成。HE染色和電鏡觀察結(jié)果表明獨(dú)活寄生湯和奧泰靈均可以促進(jìn)OA軟骨的修復(fù),促進(jìn)軟骨細(xì)胞增殖,兩者無(wú)明顯差別。獨(dú)活寄生湯和奧泰靈均能促進(jìn)Cyclin D1、CDK4、Rb mRNA和蛋白,抑制p16、Cytochrome c、 caspase-9及caspase-3mRNA和蛋白的表達(dá),兩者無(wú)顯著性差異。 2.第3代軟骨細(xì)胞Ⅱ型膠原免疫組織化學(xué)染色,軟骨細(xì)胞胞漿被染成棕黃色,胞核不著色；阿利新藍(lán)染色,軟骨細(xì)胞核為淺藍(lán)色,細(xì)胞質(zhì)中可見(jiàn)不少淺藍(lán)色的分泌顆粒和小泡。獨(dú)活寄生湯含藥血清干預(yù)軟骨細(xì)胞增殖最佳采血時(shí)間點(diǎn)為2小時(shí),最佳干預(yù)濃度為10%。采用10ng/ml IL-1β誘導(dǎo)的軟骨細(xì)胞,與正常細(xì)胞相比,細(xì)胞體積變大,邊緣出現(xiàn)一些指狀突起,核也變大,胞膜和胞漿不清晰,細(xì)胞呈多邊形改變,折光度下降；Ⅱ型膠原免疫組化顯示軟骨細(xì)胞胞漿棕黃色染色變淺。含藥血清干預(yù)后,退變軟骨細(xì)胞G0/G1期比例逐漸降低,S期比例和增殖指數(shù)逐漸增加；退變軟骨細(xì)胞的凋亡率逐漸降低；線粒體膜電位逐漸降低；隨著干預(yù)時(shí)間的延長(zhǎng),caspase-9和caspase-3活性均逐漸降低；含藥血清能促進(jìn)Cyclin D1、CDK4、Rb、Bcl-2mRNA和蛋白表達(dá),抑制p16和Bax mRNA和蛋白的表達(dá)。 結(jié)論 1.體內(nèi)外研究表明,獨(dú)活寄生湯通過(guò)促進(jìn)軟骨細(xì)胞周期關(guān)鍵限制點(diǎn)G1/S期的切換,促進(jìn)軟骨細(xì)胞增殖。 2.體內(nèi)外研究表明,獨(dú)活寄生湯通過(guò)抑制線粒體凋亡通路的激活,抑制軟骨細(xì)胞凋亡。
[Abstract]:Purpose

Objective To investigate the effect of live parasitic soup on the proliferation and apoptosis of articular chondrocytes in rats with osteoarthritis .

method

One week , 144 male SD rats were randomly divided into two groups : blank group ( 36 rats ) and model group ( 108 only ) . The model group was randomly divided into model group ( 36 rats ) , treatment group ( 36 rats ) and positive group ( 36 rats ) . The blank group and the model group were administered 0.9 % physiological saline in 10 ml 路 kg - 1 路 d -1 .
the treatment group is administered with a live parasitic soup according to the dosage of 9.3 g 路 kg - 1 路 d - 1 ;
The positive group was administered in the amount of 0.15 g - kg - 1d - 1 to the Oteling capsule ;
1 course of treatment every 2 weeks , 2 days in the middle and 4 courses of treatment .

2 . After every 2 treatment courses , a group of experimental animals were sacrificed , the inner cartilage of the tibial plateau was cut and fixed in 4 % polyformaldehyde , paraffin embedded and HE staining was used to observe the morphological changes of cartilage tissue ;
The expression of Cyclin D1 , Rb , p16 , Bc1 - 2 , Bax , bcl c , caspase - 9 and caspase - 3 protein were detected by immunohistochemistry .
The outer cartilage of the tibial plateau was excised and fixed in the electron microscope fixing solution , which was used to observe the ultrastructural changes of the chondrocytes .
The remaining cartilage tissues were rapidly placed in liquid nitrogen and used to detect the expression of Cyclin D1 , cd4 , Rb and p16mRNA .

3 . 144 male and female male SD rats were randomly divided into two groups : serum control group ( blank serum group , 36 rats ) and single live parasitic soup ( including serum group , 108 ) . the blank serum group was given 0.9 % physiological saline in the amount of 10ml 路 kg - 1 路 d - 1 ;
The drug - containing serum group was administered in a dose of 9.3 g - kg - 1 - d - 1 for 7 consecutive days . After the last 1 day for 2 consecutive times of gavage , the middle interval was 2 h , the blood was fasted for 12 h before blood sampling . After the last dose of 1 h , 2 h and 3 h , the blood was collected from the aorta of the lower abdominal aorta with 2 % sodium opental 2 ml / kg , and the serum was prepared .

4 . In healthy 4 - week - old SD rats , 20 - 30 healthy SD rats were cultured for 24 h in DMEM medium containing 10 % FBS for 24 h , and cultured for 24 h in DMEM medium containing 10 % FBS . After 24 h , 36 h , 48 h , 60 h and 72 h , the activity of chondrocytes was detected by MTT assay .

5 . Using 10 ng / ml IL - 1尾 to induce the replication and degeneration of chondrocytes in the third generation of chondrocytes , the morphological changes of cells and the immunohistochemical staining of type 鈪，

本文編號(hào)：2135308