【摘要】：目的:通過(guò)觀察六味地黃丸對(duì)兔椎間盤(pán)體外退變模型椎間盤(pán)細(xì)胞MAPK信號(hào)級(jí)聯(lián)的影響,探討六味地黃丸防治椎間盤(pán)退變的療效。方法:含藥血清制備:6月齡清潔級(jí)新西蘭白兔5只,體重2.0-3.2kg之間,湖南中醫(yī)藥大學(xué)動(dòng)物中心提供,以六味地黃丸懸液灌胃,按人和動(dòng)物體表面積比計(jì)算動(dòng)物等效給藥量給藥,空白對(duì)照組灌以等量生理鹽水。每天兩次,共7天,自然喂養(yǎng),標(biāo)準(zhǔn)飼養(yǎng)環(huán)境。在最后一次灌胃4小時(shí)后,經(jīng)心臟穿刺采血,離心、過(guò)濾、滅活后備用。選取6月齡清潔級(jí)新西蘭兔20只,參照Haschtmann方法建立整體椎間盤(pán)體外培養(yǎng)體系,空氣栓塞法處死動(dòng)物,無(wú)菌條件下采用后正中入路或經(jīng)腹腔前入路取出帶終板的L1-L6椎間盤(pán)100只,盡量去除終板上附著的骨組織及韌帶,保留終板軟骨的完整性,連續(xù)以0.9%Na Cl溶液,聚烯吡酮磺溶液以及含55 mmol/L檸檬酸鈉、50μg/m L慶大霉素的Hanks液漂洗。椎間盤(pán)樣本置于六孔板中,放在轉(zhuǎn)鼓上(轉(zhuǎn)鼓的轉(zhuǎn)速為10r?h-1),以含5%胎牛血清、50μg/m L慶大霉素、20mmol/L檸檬酸鈉的DMEM/F12培養(yǎng),置于37℃、5%CO2、100%相對(duì)濕度溫箱中過(guò)夜。隨機(jī)分為空白對(duì)照組,TNF-α組、p38-JNK/SAPK阻斷組,六味地黃丸血清組、TNF-α+含六味地黃丸血清組,將過(guò)夜后標(biāo)本接種于六孔板,加入8ml含10%胎牛血清、50μg/m L慶大霉素、25μg/m L維生素C的DMEM/F12,置于37℃、5%CO2、100%相對(duì)濕度溫箱中培養(yǎng),每?jī)商鞊Q液一次。TNF-α組培養(yǎng)液中另含10ng/μl TNF-α2μl;p38-JNK阻斷組另含p38MAPK特異性阻斷劑SB203580,JNK特異性阻斷劑SP600125各20μmol/L各10μl,10ng/μl TNF-α2μl,六味地黃丸血清組培養(yǎng)液中另含六味地黃丸血清10%;TNF-α+含六味地黃丸血清組含10ng/μl TNF-α2μl,六味地黃丸血清10%。分別于培養(yǎng)的第2、4、8、14天收集標(biāo)本待測(cè)。結(jié)果:同一時(shí)間點(diǎn)的組間比較,1.TNF-α組與空白組比較,有顯著差異P0.05,TNF-α組的JNK及P38通路的m RNA含量、蛋白表達(dá)量均明顯高于空白組,在光鏡下纖維環(huán)結(jié)構(gòu)的裂隙明顯增加,髓核細(xì)胞的形態(tài)變化明顯。說(shuō)明椎間盤(pán)已進(jìn)入退變過(guò)程,造模成功。2.P38.JNK阻斷劑組與TNF-α+六味地黃丸血清組比較,P0.05,P38.JNK阻斷劑組的JNK及P38通路的m RNA含量、蛋白表達(dá)量均明顯低于TNF-α+六味地黃丸血清組。TNF-α+六味地黃丸血清組與TNF-α組比較,P0.05,有明顯差異。TNF-α+六味地黃丸血清組的JNK及P38通路的m RNA含量、蛋白表達(dá)量均明顯低于TNF-α組,可說(shuō)明六味地黃丸可一定程度阻滯JNK及P38通路激活,延緩椎間盤(pán)退變。不同時(shí)間點(diǎn)的組內(nèi)比較,1.TNF-α組的JNK及P38通路的m RNA含量、蛋白表達(dá)量在2d與4d、4d與8d、8d與14d相比有明顯差異P0.05,含量一直呈上升趨勢(shì),在光鏡下隨著時(shí)間的推移,纖維環(huán)結(jié)構(gòu)的裂隙明顯增加,髓核細(xì)胞的形態(tài)變化明顯,可說(shuō)明造模成功。2.TNF-α+六味地黃丸血清組JNK及P38通路的m RNA含量、蛋白表達(dá)量在2d與4d、4d與8d、8d與14d相比有明顯差異P0.05,表達(dá)含量呈上升趨勢(shì),但是上升趨勢(shì)明顯低于TNF-α組,也說(shuō)明了六味地黃丸可一定程度阻滯JNK及P38通路激活,延緩椎間盤(pán)退變。結(jié)論:六味地黃丸可通過(guò)一定程度的抑制兔椎間盤(pán)細(xì)胞JNK、P38通路的基因和蛋白表達(dá),減輕椎間盤(pán)細(xì)胞的炎癥反應(yīng),延緩椎間盤(pán)退變進(jìn)程,起到保護(hù)椎間盤(pán)細(xì)胞的作用。
[Abstract]:Objective: To study the effect of Liuwei Dihuang pill on the control of disc degeneration in the disc of rabbit intervertebral disc in vitro. Methods: The preparation of drug-containing serum:5 rabbits with 6-month-old clean-grade New Zealand white rabbits and 2.0-3.2kg of body weight, were supplied by the animal center of the Chinese University of Traditional Chinese Medicine in Hunan Province. The body surface area of the six-part-six-month-old New Zealand white rabbits was given by gavage, and the equivalent dose of the animals was calculated according to the body surface area of the human and the animals. The blank control group was given the same amount of normal saline. Twice daily for 7 days, natural feeding, standard feeding environment. After the last gastric administration for 4 hours, the blood was collected by the heart, centrifuged, filtered and inactivated for backup. 20 of the six-month-old clean-grade New Zealand rabbits were selected, the whole-disc in-vitro culture system was established with reference to the Haschtmann method, the animals were sacrificed by the air embolism method, The bone tissue and ligament attached to the endplates were removed as much as possible, the integrity of the end plate cartilage was preserved, and the Hanks solution containing 55 mmol/ L sodium citrate and 50. m u.g/ m L of gentamicin was continuously rinsed with a 0.9% Na Cl solution, a polyalkenylone sulfonate solution, and a 55 mmol/ L sodium citrate, 50.mu. g/ m L Gentamicin. The disc sample was placed in a six-well plate, placed on a drum (the rotational speed of the drum was 10 r? h-1), cultured in DMEM/ F12 containing 5% fetal bovine serum,50 & mu; g/ m L of gentamicin, and 20 mmol/ L sodium citrate, and was placed overnight at 37.degree. C.,5% CO2, and 100% relative humidity. and randomly divided into a blank control group, a TNF-antigen group, a p38-JNK/ SAPK blocking group, a six-flavor dihuang pill serum group, a TNF-1 + containing Liuwei Dihuang pill serum group, an overnight sample is inoculated on a six-well plate,8 ml of DMEM/ F12 containing 10% fetal bovine serum,50 & mu; g/ m L of gentamicin and 25 & mu; g/ m L of vitamin C is added, and is placed at 37 & deg; C; Culture in 5% CO2 and 100% relative humidity incubator, and once every two days. The other contains 10 ng/. mu.l of TNF-HC2.mu. l in the culture solution of TNF-JNK, and the other p38MAPK-specific blocking agent SB203580 and JNK-specific blocking agent SP600125 in the p38-JNK blocking group contains 10. mu. l,10 ng/ mu. l of TNF-2. mu. l, and the other six-wei Dihuang pill serum is 10% in the serum group culture solution of the Liuwei Dihuang Pills. The serum group of TNF-1 + containing Liuwei Dihuang pill contains 10 ng/. mu.l of TNF-2. mu. l, and the serum of Liuwei Dihuang pill is 10%. The specimens were collected on day 2,4,8 and 14 of culture, respectively. Results: Compared with the blank group, the amount of mRNA and protein expression of the JNK and P38 in the TNF-linked group were significantly higher than that of the blank group, and the fracture of the fiber ring structure was significantly increased under the light microscope. The morphological changes of the nucleus pulposus cells were significant. The results showed that the expression of mRNA and protein of JNK and P38 in the group of JNK and P38 in the group of P38. JNK blocker group and TNF-1 + Liuwei Dihuang pill group were significantly lower than that in the serum group of TNF-1 + Liuwei Dihuang pill. The serum group of TNF-1 + Liuwei Dihuang pill group was compared with that of TNF-1 group, P 0.05, and there was a significant difference. The expression of mRNA and protein of JNK and P38 in the serum of TNF-1 + Liuwei Dihuang Pills was significantly lower than that of the TNF-1 group, and the activation of JNK and P38 pathway could be blocked to a certain extent, and the degeneration of the disc was delayed. The expression of m-RNA of JNK and P38 in TNF-1 group was significantly different between 2d and 4d, 4d, 8d, 8d and 14d. 2. The expression of m-RNA of JNK and P38 in the serum group of TNF-1 + 6-wei Dihuang Pills was significantly different from that of the 4-d,4-d, and 8d, 8d, and 14d, and the expression level was on the rise. However, that increase trend is obviously lower than that of the TNF-1 group, and the six-wei-dihuang pill can block the activation of JNK and P38 via a certain degree, and delay the degeneration of the disc. Conclusion: Liuwei Dihuang Wan can inhibit the expression of the gene and protein of the JNK and P38 pathway of the rabbit intervertebral disc cells to a certain extent, reduce the inflammatory reaction of the intervertebral disc cells, delay the disc degeneration, and play a role in protecting the intervertebral disc cells.
【學(xué)位授予單位】：湖南中醫(yī)藥大學(xué)
【學(xué)位級(jí)別】：碩士
【學(xué)位授予年份】：2016
【分類號(hào)】：R285.5;R-332

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