VDR及EMP-1在人退變腰椎間盤髓核組織中的表達(dá)研究
發(fā)布時(shí)間：2018-08-01 12:32
【摘要】：目的:通過免疫組織化學(xué)及實(shí)時(shí)熒光定量PCR等方法檢測(cè)維生素D受體(vitamin D receptor,VDR)及上皮膜蛋白1(Epithelial membrane protein1,EMP-1)在人退變腰椎間盤髓核組織中的表達(dá),探討在人類腰椎間盤髓核組織中表達(dá)變化及在退變中的作用機(jī)制,為預(yù)防及治療椎間盤退變引起的椎間盤退變性疾病提供新的思路。方法:(1).取材均來自川北醫(yī)學(xué)院附屬醫(yī)院骨科2016年1月—2016年8月的23例手術(shù)后腰椎間盤髓核標(biāo)本,分為對(duì)照組和實(shí)驗(yàn)組。對(duì)照組診斷為腰椎爆裂性骨折并行前路手術(shù),共8例,其中男性5例,女性3例,平均年齡28.38±6.84歲,既往健康,排除其他慢性疾病,經(jīng)影像學(xué)證實(shí)無明顯椎間盤退變(Pfirrmann分級(jí)標(biāo)準(zhǔn)分級(jí)為I~II級(jí))。實(shí)驗(yàn)組診斷為腰椎間盤突出癥患者,共15例,其中男性10例,女性5例,平均年齡43.60±5.82歲,既往健康,排除其他慢性疾病,經(jīng)影像學(xué)證實(shí)存在明顯椎間盤退變(Pfirrmann分級(jí)標(biāo)準(zhǔn)分級(jí)為III~Ⅴ級(jí)),且有明顯腰腿痛患者。以上均通過倫理委員會(huì),患者本人及家屬同意病簽字。(2).同一患者的腰椎間盤髓核組織分為兩塊,一塊用4%中性甲醛固定,常規(guī)脫水石蠟包埋,待HE染色和免疫組織化學(xué)檢查。另一塊椎間盤髓核組織保存在凍存管內(nèi)存放于-80°C冰箱,后期行熒光定量PCR檢測(cè)。實(shí)驗(yàn)數(shù)據(jù)應(yīng)用SPSS 19.0統(tǒng)計(jì)軟件處理,統(tǒng)計(jì)作圖應(yīng)用GraphPad Prism 6.0軟件。計(jì)量資料采用均數(shù)標(biāo)準(zhǔn)差(sx±)表示,兩樣本均數(shù)的比較采用t檢驗(yàn),P0.05為差異具有統(tǒng)計(jì)學(xué)意義,計(jì)數(shù)資料采用率進(jìn)行描述,統(tǒng)計(jì)推斷采用χ2檢驗(yàn)或Fisher確切概率法,P0.05為差異具有統(tǒng)計(jì)學(xué)意義。結(jié)果:(1).he染色及髓核細(xì)胞計(jì)數(shù):對(duì)照組髓核組織在he染色切片中表現(xiàn)為髓核細(xì)胞分布較均勻,細(xì)胞呈卵圓形,細(xì)胞周圍有不規(guī)則的陷窩,細(xì)胞多孤立存在于細(xì)胞陷窩內(nèi)。實(shí)驗(yàn)組髓核細(xì)胞胞質(zhì)中可見大量空泡,髓核細(xì)胞肥大,多個(gè)肥大的髓核細(xì)胞聚集在一起,呈現(xiàn)髓核細(xì)胞的“巢狀”結(jié)構(gòu)。髓核細(xì)胞在基質(zhì)中分布很不均勻,主要表現(xiàn)為呈“巢狀”結(jié)構(gòu)的髓核細(xì)胞團(tuán)多出現(xiàn)在椎間盤裂痕的周圍。對(duì)髓核細(xì)胞進(jìn)行計(jì)數(shù),對(duì)照組髓核細(xì)胞平均數(shù)為:174.58±10.23(個(gè)/單位視野100x),實(shí)驗(yàn)組為:88.64±3.16(個(gè)/單位視野100x),對(duì)照組與實(shí)驗(yàn)組髓核組織中的髓核細(xì)胞數(shù)相比,實(shí)驗(yàn)組中髓核細(xì)胞數(shù)較對(duì)照組明顯減少,差異有統(tǒng)計(jì)學(xué)意義(p0.05)。(2).免疫組織化學(xué):測(cè)得vdr在對(duì)照組和實(shí)驗(yàn)組陽性細(xì)胞率分別為25%和86.67%;emp-1在對(duì)照組和實(shí)驗(yàn)組陽性細(xì)胞率分別為12.5%和93.33%;結(jié)果表明實(shí)驗(yàn)組中vdr和emp-1的表達(dá)情況明顯強(qiáng)于對(duì)照組(p0.05)。(3).實(shí)時(shí)熒光定量pcr法檢測(cè):可見各椎間盤髓核組織標(biāo)本中的基因擴(kuò)增曲線、熔解曲線良好,未出現(xiàn)明顯雜峰和主峰的異常增寬。vdr和emp-1mrna在實(shí)驗(yàn)組中的表達(dá)均明顯高于對(duì)照組(p0.05)。結(jié)論:(1).人退變腰椎間盤髓核組織中髓核細(xì)胞數(shù)量明顯減少;(2).退變腰椎間盤髓核組織中vdr及emp-1的表達(dá)明顯升高;(3).腰椎間盤髓核組織中vdr及emp-1表達(dá)的升高可能是導(dǎo)致退變腰椎間盤中髓核細(xì)胞增殖與凋亡失衡的原因之一,同時(shí)也是引起腰椎間盤退變的重要機(jī)制之一。
[Abstract]:Objective: to detect the expression of vitamin D receptor (vitamin D receptor (VDR) and upper membrane protein 1 (Epithelial membrane protein1, EMP-1) in the lumbar intervertebral disc nucleus of human degeneration by immunohistochemistry and real-time fluorescence quantitative PCR, and to explore the expression changes in the human lumbar intervertebral disc nucleus and the mechanism of action in the degeneration of human lumbar intervertebral disc. To provide new ideas for the prevention and treatment of degenerative disc degeneration caused by intervertebral disc degeneration. Methods: (1) 23 cases of lumbar disc nucleus pulposus from the Department of orthopedics of Affiliated Hospital of Chuanbei Medical College from January 2016 to August 2016 were collected and divided into the control group and the experimental group. The control group was diagnosed as a parallel anterior approach to the burst fracture of the lumbar spine. A total of 8 cases, including 5 male and 3 female, with an average age of 28.38 + 6.84 years old, were previously healthy and excluded other chronic diseases. There were no obvious intervertebral disc degeneration (Pfirrmann grading standard I~II). The experimental group was diagnosed as lumbar intervertebral disc herniation, with 15 cases, including 10 men and 5 women, with an average age of 43.60 + 5.82 years old. To health and to eliminate other chronic diseases, it was confirmed by imaging that there was an obvious disc degeneration (Pfirrmann grading standard III~ V), and patients with lumbago and leg pain were obvious. All of these were approved by the ethics committee, the patient and the family agreed to sign the disease. (2) the lumbar disc nucleus of the same patient was divided into two pieces, one with 4% neutral formaldehyde. Fixed, dehydrated paraffin embedded, HE staining and immunohistochemical examination. The other intervertebral disc nucleus was stored in the cryopreservation tube in -80 / C refrigerator, and the fluorescence quantitative PCR was detected at the later stage. The experimental data were treated with SPSS 19 statistical software, and the statistical mapping was applied to the GraphPad Prism 6 software. The measurement data used the mean standard deviation (s). X +) indicated that the comparison of the average number of two samples was compared with t test. The difference was statistically significant in P0.05, and the use rate of the count data was described. The statistical inference was made by x 2 test or Fisher exact probability. The difference was statistically significant. Results: (1).He staining and nucleus pulposus count: the control group was displayed in HE staining section. The nucleus pulposus cells are evenly distributed, the cells are oval, and there are irregular lacunae around the cells. The cells are mostly isolated in the cell lacunae. In the experimental group, a large number of vacuoles are found in the cytoplasm of the nucleus pulmedulla cells, the nucleus cells of the medullary nucleus are hypertrophic, and the nucleus cells of the nucleus pulmedes are gathered together, and the nucleus pulposus cells are divided into the matrix. The distribution of the nucleus pulmedullary cell mass appeared mostly around the disc fissure. The nucleus pulposus cells were counted. The average number of nucleus pulmedullary cells in the control group was 174.58 + 10.23 (100x per unit field of vision), the experimental group was 88.64 + 3.16 (100x / single visual field), the control group and the nucleus pulposus nucleus of the experimental group. Compared with the control group, the number of nucleus pulposus cells in the experimental group was significantly lower than that in the control group (P0.05). (2). Immunohistochemistry: the positive cell rates of VDR in the control and experimental groups were 25% and 86.67%, respectively, and the positive cell rates of EMP-1 in the control and experimental groups were 12.5% and 93.33%, respectively. The results showed that in the experimental group, the VDR and EMP-1 were in the experimental group. The expression was obviously stronger than that of the control group (P0.05). (3). Real time fluorescence quantitative PCR method was used to detect the gene amplification curve in the tissue specimens of intervertebral discs. The fusion curve was good, the abnormal widening of the main peaks and the abnormal widening of.Vdr and emp-1mrna in the experimental group were obviously higher than those of the control group (P0.05). Conclusion: (1) the degenerative lumbar vertebra The number of nucleus pulmedullary cells in the nucleus pulposus of intervertebral disc decreased significantly, (2) the expression of VDR and EMP-1 in the degenerative lumbar intervertebral disc increased obviously; (3) the increase of VDR and EMP-1 in the lumbar intervertebral disc nucleus may be one of the causes of the imbalance of the proliferation and apoptosis of the nucleus pulmedulo cells in the degenerative lumbar intervertebral disc, and also the cause of the lumbar intervertebral disc degeneration. One of the important mechanisms of change.
【學(xué)位授予單位】：川北醫(yī)學(xué)院
【學(xué)位級(jí)別】：碩士
【學(xué)位授予年份】：2017
【分類號(hào)】：R681.53
本文編號(hào)：2157591
[Abstract]:Objective: to detect the expression of vitamin D receptor (vitamin D receptor (VDR) and upper membrane protein 1 (Epithelial membrane protein1, EMP-1) in the lumbar intervertebral disc nucleus of human degeneration by immunohistochemistry and real-time fluorescence quantitative PCR, and to explore the expression changes in the human lumbar intervertebral disc nucleus and the mechanism of action in the degeneration of human lumbar intervertebral disc. To provide new ideas for the prevention and treatment of degenerative disc degeneration caused by intervertebral disc degeneration. Methods: (1) 23 cases of lumbar disc nucleus pulposus from the Department of orthopedics of Affiliated Hospital of Chuanbei Medical College from January 2016 to August 2016 were collected and divided into the control group and the experimental group. The control group was diagnosed as a parallel anterior approach to the burst fracture of the lumbar spine. A total of 8 cases, including 5 male and 3 female, with an average age of 28.38 + 6.84 years old, were previously healthy and excluded other chronic diseases. There were no obvious intervertebral disc degeneration (Pfirrmann grading standard I~II). The experimental group was diagnosed as lumbar intervertebral disc herniation, with 15 cases, including 10 men and 5 women, with an average age of 43.60 + 5.82 years old. To health and to eliminate other chronic diseases, it was confirmed by imaging that there was an obvious disc degeneration (Pfirrmann grading standard III~ V), and patients with lumbago and leg pain were obvious. All of these were approved by the ethics committee, the patient and the family agreed to sign the disease. (2) the lumbar disc nucleus of the same patient was divided into two pieces, one with 4% neutral formaldehyde. Fixed, dehydrated paraffin embedded, HE staining and immunohistochemical examination. The other intervertebral disc nucleus was stored in the cryopreservation tube in -80 / C refrigerator, and the fluorescence quantitative PCR was detected at the later stage. The experimental data were treated with SPSS 19 statistical software, and the statistical mapping was applied to the GraphPad Prism 6 software. The measurement data used the mean standard deviation (s). X +) indicated that the comparison of the average number of two samples was compared with t test. The difference was statistically significant in P0.05, and the use rate of the count data was described. The statistical inference was made by x 2 test or Fisher exact probability. The difference was statistically significant. Results: (1).He staining and nucleus pulposus count: the control group was displayed in HE staining section. The nucleus pulposus cells are evenly distributed, the cells are oval, and there are irregular lacunae around the cells. The cells are mostly isolated in the cell lacunae. In the experimental group, a large number of vacuoles are found in the cytoplasm of the nucleus pulmedulla cells, the nucleus cells of the medullary nucleus are hypertrophic, and the nucleus cells of the nucleus pulmedes are gathered together, and the nucleus pulposus cells are divided into the matrix. The distribution of the nucleus pulmedullary cell mass appeared mostly around the disc fissure. The nucleus pulposus cells were counted. The average number of nucleus pulmedullary cells in the control group was 174.58 + 10.23 (100x per unit field of vision), the experimental group was 88.64 + 3.16 (100x / single visual field), the control group and the nucleus pulposus nucleus of the experimental group. Compared with the control group, the number of nucleus pulposus cells in the experimental group was significantly lower than that in the control group (P0.05). (2). Immunohistochemistry: the positive cell rates of VDR in the control and experimental groups were 25% and 86.67%, respectively, and the positive cell rates of EMP-1 in the control and experimental groups were 12.5% and 93.33%, respectively. The results showed that in the experimental group, the VDR and EMP-1 were in the experimental group. The expression was obviously stronger than that of the control group (P0.05). (3). Real time fluorescence quantitative PCR method was used to detect the gene amplification curve in the tissue specimens of intervertebral discs. The fusion curve was good, the abnormal widening of the main peaks and the abnormal widening of.Vdr and emp-1mrna in the experimental group were obviously higher than those of the control group (P0.05). Conclusion: (1) the degenerative lumbar vertebra The number of nucleus pulmedullary cells in the nucleus pulposus of intervertebral disc decreased significantly, (2) the expression of VDR and EMP-1 in the degenerative lumbar intervertebral disc increased obviously; (3) the increase of VDR and EMP-1 in the lumbar intervertebral disc nucleus may be one of the causes of the imbalance of the proliferation and apoptosis of the nucleus pulmedulo cells in the degenerative lumbar intervertebral disc, and also the cause of the lumbar intervertebral disc degeneration. One of the important mechanisms of change.
【學(xué)位授予單位】：川北醫(yī)學(xué)院
【學(xué)位級(jí)別】：碩士
【學(xué)位授予年份】：2017
【分類號(hào)】：R681.53
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