本文選題：硬膜外瘢痕粘連 + 羥基喜樹(shù)堿�。� 參考：《揚(yáng)州大學(xué)》2017年碩士論文

【摘要】：目的:探討四種不同濃度梯度的羥基喜樹(shù)堿(10-Hydroxycamptothecin,HCPT)局部使用對(duì)椎板切除術(shù)后硬膜外瘢痕粘連的預(yù)防作用,并進(jìn)一步從細(xì)胞水平探究其預(yù)防硬膜外粘連的具體機(jī)制。方法:將72只Sprague-Dawley雄性大鼠平均四組,分別是0.2 mg/ml組、0.1 mg/ml組、0.05 mg/ml和生理鹽水對(duì)照組。采用1%濃度的戊巴比妥鈉對(duì)所有大鼠予腹腔注射麻醉后,進(jìn)行常規(guī)術(shù)前準(zhǔn)備后,在L1-L2附近逐層進(jìn)入后顯露椎體后面,使用微小咬骨鉗在L1椎板制造硬脊膜暴露區(qū),面積大小約5mm×2mm,然后將各種不同濃度的HCPT和生理鹽水在紗布的保護(hù)下局部應(yīng)用于人工造成的骨缺損區(qū),計(jì)時(shí)5分鐘,然后使用預(yù)先準(zhǔn)備好的無(wú)菌生理鹽水沖洗殘留藥物,在關(guān)閉切口之前再次進(jìn)行創(chuàng)口消毒,手術(shù)后連續(xù)3天使用抗生素肌肉注射治療以預(yù)防感染。28天后再次進(jìn)行手術(shù),逐層打開(kāi),對(duì)硬膜外區(qū)域行瘢痕粘連的大體評(píng)估,心臟灌注處死后,取下目標(biāo)椎體還有其周?chē)慕M織,對(duì)所有標(biāo)本實(shí)行脫鈣處理,然后使用石蠟包埋組織,行HE染色法在不同放大率的顯微鏡下觀察不同濃度HCPT對(duì)椎板切除術(shù)后粘連的預(yù)防以及局部成纖維細(xì)胞增殖抑制的效果,硬膜外瘢痕中NOXA的表達(dá)量的情況由免疫組織化學(xué)染色法測(cè)定,TUNEL檢測(cè)用來(lái)測(cè)定硬膜外瘢痕組織中成纖維細(xì)胞的凋亡率變化。體外培養(yǎng)成纖維細(xì)胞,予不同濃度的HCPT(0,1,2,4 μg/ml)刺激成纖維細(xì)胞,TUNEL染色法觀察HCPT刺激成纖維細(xì)胞后細(xì)胞凋亡率的變化,Western-blot檢測(cè)凋亡相關(guān)蛋白表達(dá)情況。予慢病毒載體敲除NOXA基因后,TUNEL染色法檢測(cè)觀察HCPT刺激后的成纖維細(xì)胞的凋亡率的變化,Western-blot檢測(cè)NOXA以及凋亡相關(guān)蛋白的表達(dá)量變化。結(jié)果:本實(shí)驗(yàn)中所有的大鼠均未發(fā)生癱瘓、感染、死亡等現(xiàn)象。HE染色結(jié)果顯示:0.2 mg/ml HCPT組、0.1 mg/ml HCPT組以及0.05 mg/ml HCPT組的瘢痕粘連程度明顯好于對(duì)照組,而高倍鏡下觀察到HCPT治療組硬膜外瘢痕組織中成纖維細(xì)胞數(shù)量明顯少于對(duì)照組硬膜外瘢痕中的成纖維細(xì)胞的數(shù)量,差異具有統(tǒng)計(jì)學(xué)意義(P0.05)。對(duì)于硬膜外瘢痕組織中成纖維細(xì)胞的TUNEL染色顯示:成纖維細(xì)胞的凋亡率也升高趨勢(shì)和HCPT的劑量趨勢(shì)呈正相關(guān)。此外,硬膜外瘢痕組織中免疫組織化學(xué)染色結(jié)果顯示HCPT治療組中的NOXA表達(dá)量明顯多于對(duì)照組中NOXA的表達(dá)量,伴隨著HCPT濃度的升高,NOXA的表達(dá)量也呈現(xiàn)升高趨勢(shì),提示NOXA的表達(dá)量的增加可能與HCPT對(duì)硬膜外瘢痕粘連的抑制作用有關(guān)。體外細(xì)胞實(shí)驗(yàn)的結(jié)果顯示HCPT能有效抑制成纖維細(xì)胞的細(xì)胞活性,TUNEL染色發(fā)現(xiàn)所有HCPT處理組的成纖維細(xì)胞凋亡率明顯多于對(duì)照組。Western-blot結(jié)果顯示應(yīng)用不同濃度的HCPT刺激成纖維細(xì)胞后,NOXA,cleaved-PARP,Bax的表達(dá)量隨著HCPT濃度的增高而升高,而B(niǎo)cl-2的表達(dá)量則成相反趨勢(shì),此外,使用慢病毒技術(shù)敲除NOXA之后,我們通過(guò)TUNEL染色發(fā)現(xiàn)成纖維細(xì)胞的凋亡率相應(yīng)的降低了,而且Western-blot結(jié)果也顯示凋亡相關(guān)蛋白的表達(dá)量也隨之降低。結(jié)論:HCPT可以有效的預(yù)防大鼠椎板切除術(shù)后硬膜外瘢痕粘連的形成,且其預(yù)防作用的潛在機(jī)制可能是通過(guò)上調(diào)NOXA的表達(dá)、促進(jìn)成纖維細(xì)胞凋亡。
[Abstract]:Objective: To explore the preventive effect of four different concentration gradient 10-Hydroxycamptothecin (HCPT) on epidural scar adhesion after laminectomy, and to further explore the specific mechanism of preventing epidural adhesion from the level of cell. Methods: the average four groups of 72 Sprague-Dawley male rats were 0.2 mg/ml, respectively. Group, 0.1 mg/ml, 0.05 mg/ml and saline control group. After intraperitoneal injection of pentobarbital, 1% concentrations of sodium pentobarbital were used for routine preoperative preparation, and then the vertebral body was exposed after layer by layer near L1-L2. Small bone nippers were used to make the dural exposed area in the L1 vertebral lamina. The size of the spinal cord was about 5mm * 2mm, and then various kinds of non - The same concentration of HCPT and normal saline were applied to the artificial bone defect area under the protection of gauze for 5 minutes, then the residual drugs were washed with pre prepared aseptic saline, and the wound was sterilized again before closing the incision. After 3 days of operation, antibiotic injection was used to prevent infection after.28 days. After the operation, it was opened by layer by layer to assess the scar adhesion in the epidural area. After the heart perfusion was executed, the target vertebral body and its surrounding tissue were removed, and all the specimens were decalcified. Then the paraffin embedded tissue was used, and the HE staining method was used to observe the different concentration of HCPT under the microscope for laminectomy under the microscope of different magnification. The prevention of posterior adhesion and the effect of local fibroblast proliferation inhibition. The expression of NOXA in epidural scar was measured by immunohistochemical staining. TUNEL detection was used to determine the apoptosis rate of fibroblasts in epidural scar tissue. In vitro culture fibroblasts were cultured with different concentrations of HCPT (0,1,2,4 mu g/ml) spines. The apoptosis rate of HCPT stimulated fibroblasts was observed by TUNEL staining, and the expression of apoptosis related proteins was detected by Western-blot. After the lentivirus vector knockout NOXA gene, the apoptosis rate of fibroblasts stimulated by HCPT was detected by TUNEL staining, Western-blot detection NOXA and apoptosis related Results: all rats in this experiment had no paralysis, infection, and death. The results of.HE staining showed that the degree of scar adhesion in 0.2 mg/ml HCPT group, 0.1 mg/ml HCPT group and 0.05 mg/ml HCPT group was better than that of the control group, and the fibroblasts in the epidural scar tissue of the HCPT treatment group were observed under the high magnification microscope. The number of cells in the epidural scar was significantly less than that in the control group. The difference was statistically significant (P0.05). The TUNEL staining of the fibroblasts in the epidural scar tissue showed that the trend of apoptosis in the fibroblasts was positively correlated with the dose trend of HCPT. In addition, the immune tissues in the epidural scar tissue were immune. The results of chemical staining showed that the expression of NOXA in the HCPT treatment group was more than the NOXA expression in the control group. With the increase of HCPT concentration, the expression of NOXA also showed an upward trend, suggesting that the increase of the expression of NOXA may be related to the inhibitory effect of HCPT on the adhesion of epidural scar. The results of cell experiments in vitro show that HCPT can be found. TUNEL staining showed that the apoptosis rate of fibroblasts in all HCPT treatment groups was significantly more than that of the control group.Western-blot results showed that the expression of NOXA, cleaved-PARP, Bax increased with the increase of HCPT concentration, and the expression of Bcl-2 was opposite to that of the control group. In addition, after using lentivirus technique to knock out NOXA, we found that the apoptosis rate of fibroblasts decreased by TUNEL staining, and the Western-blot results also showed that the expression of apoptosis related proteins also decreased. Conclusion: HCPT can effectively prevent the formation of epidural scar adhesion after laminectomy in rats, and it can be effectively prevented from the formation of the epidural scar adhesion after laminectomy in rats. The potential mechanism of preventive effect may be to promote fibroblast apoptosis by up regulating the expression of NOXA.
【學(xué)位授予單位】：揚(yáng)州大學(xué)
【學(xué)位級(jí)別】：碩士
【學(xué)位授予年份】：2017
【分類(lèi)號(hào)】：R687.3

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