頸椎突出椎間盤對后縱韌帶骨化形成機(jī)制的研究
發(fā)布時(shí)間：2018-08-27 06:03
【摘要】：實(shí)驗(yàn)背景：后縱韌帶骨化癥(ossification of the posterior longitudinal ligament, OPLL)是發(fā)生在脊柱后縱韌帶中的異位骨化,壓迫脊髓和神經(jīng)根而導(dǎo)致其功能受損的一種疾病。其發(fā)病機(jī)制至今不明確。后縱韌帶增厚及骨化與突出的椎間盤是導(dǎo)致頸椎椎管狹窄常見的原因,在臨床上,我們經(jīng)常發(fā)現(xiàn)椎間盤的突出和后縱韌帶的增厚、骨化是同時(shí)存在的,但是兩者之間是否具有相關(guān)性,目前尚未見報(bào)道。目的：本實(shí)驗(yàn)旨在：在體外,研究髓核細(xì)胞分泌的細(xì)胞因子對后縱韌帶細(xì)胞增殖、細(xì)胞毒性及成骨分化能力的影響,探討頸椎退變突出的椎間盤對后縱韌帶骨化(OPLL)發(fā)展的作用,為后縱韌帶骨化癥的診治提供新的理論依據(jù)。方法：1. 骨化后縱韌帶細(xì)胞培養(yǎng)及傳代通過影像學(xué)資料(X、CT及MRI)選取后縱韌帶骨化合并頸椎間盤突出的患者10例,通過頸椎前路椎體次全切手術(shù)獲取骨化的后縱韌帶(標(biāo)本采集經(jīng)患者和家屬同意),剔除骨化的部分,保留未骨化的韌帶,用組織塊原代培養(yǎng)法行頸椎后縱韌帶成纖維細(xì)胞體外培養(yǎng)并傳代,收集第三代細(xì)胞用于實(shí)驗(yàn)。2.髓核細(xì)胞的培養(yǎng)及傳代通過影像學(xué)資料(X、CT及MRI)選取頸椎間盤突出的患者1例,通過頸椎前路椎間盤切術(shù)獲取突出的髓核組織(標(biāo)本采集經(jīng)患者和家屬同意),利用酶解法(Ⅱ型膠原酶消化)提取髓核細(xì)胞,行原代培養(yǎng)并傳傳代,收集第三代細(xì)胞用于實(shí)驗(yàn)。3. 建立實(shí)驗(yàn)分組建立實(shí)驗(yàn)組及對照組,通過Transwell建立后縱韌帶細(xì)胞和髓核細(xì)胞共培養(yǎng)體系。實(shí)驗(yàn)組：OPLL細(xì)胞接種于Transwell六孔板下室,髓核細(xì)胞接種與Transwell六孔板上室,中間隔以孔徑為0.4μm的聚酯膜；對照組：OPLL細(xì)胞種板于普通六孔板。4.酶聯(lián)免疫法(enzyme-linked immunosorbent assay, ELISA)測細(xì)胞因子實(shí)驗(yàn)組及對照組于37℃、5% C02、飽和濕度的培養(yǎng)箱培養(yǎng)兩天后,收集實(shí)驗(yàn)組及對照組細(xì)胞培養(yǎng)液,2000rpm離心20分鐘,取上清液于EP管中待測,按ELISA試劑盒操作方法分別檢測IL-1a, IL-6、 PGE2及TNF-a含量。5. MTT比色法測細(xì)胞毒性實(shí)驗(yàn)組及對照組于37℃、5% C02、飽和濕度的培養(yǎng)箱培養(yǎng)兩天后, 加入MTT液,繼續(xù)在37℃、5% C02、飽和濕度的培養(yǎng)箱培養(yǎng)4小時(shí),小心吸去每孔內(nèi)的培養(yǎng)液, 加入DMSO,酶標(biāo)儀測OD490值。6. EdU法測細(xì)胞增殖實(shí)驗(yàn)組和對照組于37℃、5% C02、飽和濕度的培養(yǎng)箱培養(yǎng)2天后,吸除培養(yǎng)液,加入含EdU溶液的培養(yǎng)基(1000：1的比例稀釋),行EdU標(biāo)記,在孵箱中繼續(xù)培養(yǎng)4小時(shí)。吸除培養(yǎng)基,磷酸鹽緩沖液清洗3遍,固定液固定細(xì)胞,依次用Apollo染色液及Hoechst染料對DNA染色。于熒光顯微鏡下鏡檢,計(jì)數(shù)細(xì)胞增殖率(紅色細(xì)胞核數(shù)／總細(xì)胞核數(shù))。7. RT-PCR法檢測mRNA的表達(dá)使用Trizol提取總RNA;用逆轉(zhuǎn)錄試劑盒反應(yīng)體系將所得RNA轉(zhuǎn)錄成cDNA,此過程分兩步進(jìn)行：首先去除基因組DNA,其次進(jìn)行反轉(zhuǎn)錄反應(yīng);將cDNA加入PCR反應(yīng)體系,進(jìn)行PCR擴(kuò)增,使用2-△△Ct值分析mRNA相對表達(dá)。8. Von Kossa染色法及ALP染色法檢測成骨活性實(shí)驗(yàn)組和對照組于37℃、5% C02及飽和濕度的培養(yǎng)箱培養(yǎng)48h后,采用ALP染色試劑盒(鈣鈷法),分別對實(shí)驗(yàn)組和對照組OPLL細(xì)胞進(jìn)行堿性磷酸酶染色。實(shí)驗(yàn)組和對照組于37℃、5% C02及飽和濕度的培養(yǎng)箱培養(yǎng)48h后,采用Von Kossa染色試劑盒操作方法對實(shí)驗(yàn)組和對照組染色。9. 統(tǒng)計(jì)分析應(yīng)用SPSS 11.5統(tǒng)計(jì)軟件對數(shù)據(jù)進(jìn)行分析,用t檢驗(yàn)方法比較組間不同處理的差異,P0.05為統(tǒng)計(jì)具有差異性。結(jié)果：1.細(xì)胞毒性MTT法檢測共培養(yǎng)體系對后縱韌帶細(xì)胞細(xì)胞毒性發(fā)現(xiàn)：實(shí)驗(yàn)組OD490均較對照組OD490高,對照組OD49o值為0.25±0.06,實(shí)驗(yàn)組OD49o為0.28±0.09,差異有統(tǒng)計(jì)學(xué)意義(P0.05)。2.細(xì)胞增殖 熒光顯微鏡下熒光激發(fā)、拍攝及合成圖片示：實(shí)驗(yàn)組中伴有DNA新合成的紅色細(xì)胞核數(shù)明顯多于對照組,在4小時(shí)內(nèi),實(shí)驗(yàn)組中OPLL細(xì)胞增殖率為14.30±2.67%,對照組中OPLL細(xì)胞增殖率為2.21±0.64%,差異有統(tǒng)計(jì)學(xué)意義(P0.05)。3. Von Kossa染色及ALP染色在Von Kossa染色中,實(shí)驗(yàn)組中有明顯的鈣結(jié)節(jié)沉淀,而對照組中鈣結(jié)節(jié)沉淀幾乎不可見；A LP染色中,實(shí)驗(yàn)組中染色陽性的細(xì)胞數(shù)明顯較對照組多。4. RT-PCR檢測Ⅰ、Ⅺ型膠原及骨鈣素mRNA的表達(dá)與對照組相比,實(shí)驗(yàn)組中Ⅰ型膠原、Ⅺ型膠原及骨鈣素mRNA的表達(dá)均上調(diào),差異具有統(tǒng)計(jì)學(xué)意義(P0.05)。5. ELISA法測炎癥因子與OPLL組及NP組相比,OPLL+NP組上清液中IL-1a 、 TNF-a 、 PGE2的含量明顯增加,差異有統(tǒng)計(jì)學(xué)意義(P0.05)。各組上清液中IL-6含量低于最低檢測標(biāo)準(zhǔn)6.25pg/ml。結(jié)論：在體外,髓核細(xì)胞能夠促進(jìn)OPLL細(xì)胞的增殖,提高OPLL細(xì)胞的成骨分化能力并且無明顯的細(xì)胞毒性。頸椎突出椎間盤促進(jìn)后縱韌帶骨化的發(fā)展,髓核組織分泌的細(xì)胞因子對后縱韌帶骨化起了重要作用。
[Abstract]:BACKGROUND: Ossification of the posterior longitudinal ligament (OPLL) is a disease that occurs in the posterior longitudinal ligament of the spine, which is heterotopic ossification, compressing the spinal cord and nerve roots and causing impairment of its function. The pathogenesis of OPLL is still unclear. Objective: To study the effect of cytokines secreted by nucleus pulposus cells on the proliferation and fineness of posterior longitudinal ligament cells in vitro. Objective: To investigate the effect of cervical intervertebral disc degeneration and protrusion on the development of ossification of posterior longitudinal ligament (OPLL) and provide new theoretical basis for the diagnosis and treatment of OPLL. Methods: 1. Cell culture and passage of ossified posterior longitudinal ligament (POL) were studied by X-ray, CT and MRI. Ten patients with cervical disc herniation underwent anterior subtotal resection of the cervical vertebra to obtain ossified posterior longitudinal ligament (specimens were collected with the consent of the patient and his family members). The ossified part was removed and the non-ossified ligament was retained. The fibroblasts of the cervical posterior longitudinal ligament were cultured and subcultured in vitro with tissue block primary culture method. The third generation cells were collected and used in the experiment. 1 patient with cervical disc herniation was selected by X-ray, CT and MRI. The prominent nucleus pulposus tissue was obtained by anterior cervical discectomy (with the consent of the patient and his family members). The nucleus pulposus cells were extracted by enzymatic hydrolysis (collagenase type II digestion). The nucleus pulposus pulposus cells were primary cultured and subcultured. The experimental group: OPLL cells were inoculated in the subcompartment of Transwell six-hole plate, the nucleus pulposus cells were inoculated in the subcompartment of Transwell six-hole plate, and the polyester membrane with a pore size of 0.4 micron was used in the middle of the compartment. LL cell culture medium was collected from the experimental group and the control group after two days of incubation in a saturated humidity incubator. The supernatant was centrifuged for 20 minutes at 2 000 rpm. The supernatant was collected and tested in EP tube. METHODS IL-1a, IL-6, PGE2 and TNF-a contents were measured by MTT colorimetric assay. The cytotoxicity of the experimental group and the control group was measured by MTT colorimetric assay at 37 C, 5% C02. After two days of incubation in saturated humidity incubator, MTT solution was added, and then incubated in 37 C, 5% C02, saturated humidity incubator for 4 hours. The culture medium in each hole was carefully sucked away, DMSO was added, and OD490 was measured by enzyme-labeling apparatus. EdU method was used to measure cell proliferation in the experimental group and the control group at 37, 5% C02, saturated humidity incubator for 2 days, then the culture medium was sucked, the medium containing EdU solution was added (1000:1 diluted), and the cells were labeled with EdU for 4 hours in the incubator. DNA was stained with LLO staining solution and Hoechst dye. Cell proliferation rate (red cell nucleus / total cell nucleus) was counted under fluorescence microscope. 7. Total RNA was extracted by Trizol and transcribed into cDNA by reverse transcription kit reaction system. The process was carried out in two steps: first, genomic DNA was removed, and the total RNA was extracted. 8. Von Kossa staining and ALP staining were used to detect the osteogenic activity of the experimental group and the control group. After incubated at 37 C, 5% C02 and saturated humidity for 48 hours, ALP staining kit (Ca-Co method) was used for the experimental group and the control group, respectively. The OPLL cells in the control group were stained with alkaline phosphatase. The experimental group and the control group were cultured at 37 C, 5% C02 and saturated humidity incubator for 48 hours. Von Kossa staining kit was used to stain the OPLL cells in the experimental group and the control group. 9. Results: 1. The cytotoxicity of the co-culture system to the posterior longitudinal ligament cells was detected by cytotoxicity MTT assay. The results showed that the OD490 of the experimental group was higher than that of the control group, and the OD49o of the control group was 0.25 [0.06], and the OD49o of the experimental group was 0.28 [0.09], the difference was statistically significant (P 0.05). 2. The number of newly synthesized red cell nuclei with DNA in the experimental group was significantly higher than that in the control group. Within 4 hours, the proliferation rate of OPLL cells in the experimental group was 14.30 (+ 2.67%) and that of OPLL cells in the control group was 2.21 (+ 0.64%). The difference was statistically significant (P 0.05). 3. Von Kossa staining and ALP staining were detected in Von Kossa staining. The number of positive cells in the experimental group was significantly higher than that in the control group. 4. The expression of collagen type I, collagen type VII and osteocalcin mRNA in the experimental group was significantly higher than that in the control group. Compared with OPLL group and NP group, the levels of IL-1a, TNF-a and PGE2 in the supernatant of OPLL+NP group were significantly increased (P 0.05). The levels of IL-6 in the supernatant of each group were lower than the minimum detection standard of 6.25 pg/ml. Conclusion: In vitro, nucleus pulposus cells can be detected. The cervical disc herniation promotes the ossification of the posterior longitudinal ligament, and the cytokines secreted by the nucleus pulposus play an important role in the ossification of the posterior longitudinal ligament.
【學(xué)位授予單位】：山東大學(xué)
【學(xué)位級別】：碩士
【學(xué)位授予年份】：2016
【分類號】：R681.5
本文編號：2206340
[Abstract]:BACKGROUND: Ossification of the posterior longitudinal ligament (OPLL) is a disease that occurs in the posterior longitudinal ligament of the spine, which is heterotopic ossification, compressing the spinal cord and nerve roots and causing impairment of its function. The pathogenesis of OPLL is still unclear. Objective: To study the effect of cytokines secreted by nucleus pulposus cells on the proliferation and fineness of posterior longitudinal ligament cells in vitro. Objective: To investigate the effect of cervical intervertebral disc degeneration and protrusion on the development of ossification of posterior longitudinal ligament (OPLL) and provide new theoretical basis for the diagnosis and treatment of OPLL. Methods: 1. Cell culture and passage of ossified posterior longitudinal ligament (POL) were studied by X-ray, CT and MRI. Ten patients with cervical disc herniation underwent anterior subtotal resection of the cervical vertebra to obtain ossified posterior longitudinal ligament (specimens were collected with the consent of the patient and his family members). The ossified part was removed and the non-ossified ligament was retained. The fibroblasts of the cervical posterior longitudinal ligament were cultured and subcultured in vitro with tissue block primary culture method. The third generation cells were collected and used in the experiment. 1 patient with cervical disc herniation was selected by X-ray, CT and MRI. The prominent nucleus pulposus tissue was obtained by anterior cervical discectomy (with the consent of the patient and his family members). The nucleus pulposus cells were extracted by enzymatic hydrolysis (collagenase type II digestion). The nucleus pulposus pulposus cells were primary cultured and subcultured. The experimental group: OPLL cells were inoculated in the subcompartment of Transwell six-hole plate, the nucleus pulposus cells were inoculated in the subcompartment of Transwell six-hole plate, and the polyester membrane with a pore size of 0.4 micron was used in the middle of the compartment. LL cell culture medium was collected from the experimental group and the control group after two days of incubation in a saturated humidity incubator. The supernatant was centrifuged for 20 minutes at 2 000 rpm. The supernatant was collected and tested in EP tube. METHODS IL-1a, IL-6, PGE2 and TNF-a contents were measured by MTT colorimetric assay. The cytotoxicity of the experimental group and the control group was measured by MTT colorimetric assay at 37 C, 5% C02. After two days of incubation in saturated humidity incubator, MTT solution was added, and then incubated in 37 C, 5% C02, saturated humidity incubator for 4 hours. The culture medium in each hole was carefully sucked away, DMSO was added, and OD490 was measured by enzyme-labeling apparatus. EdU method was used to measure cell proliferation in the experimental group and the control group at 37, 5% C02, saturated humidity incubator for 2 days, then the culture medium was sucked, the medium containing EdU solution was added (1000:1 diluted), and the cells were labeled with EdU for 4 hours in the incubator. DNA was stained with LLO staining solution and Hoechst dye. Cell proliferation rate (red cell nucleus / total cell nucleus) was counted under fluorescence microscope. 7. Total RNA was extracted by Trizol and transcribed into cDNA by reverse transcription kit reaction system. The process was carried out in two steps: first, genomic DNA was removed, and the total RNA was extracted. 8. Von Kossa staining and ALP staining were used to detect the osteogenic activity of the experimental group and the control group. After incubated at 37 C, 5% C02 and saturated humidity for 48 hours, ALP staining kit (Ca-Co method) was used for the experimental group and the control group, respectively. The OPLL cells in the control group were stained with alkaline phosphatase. The experimental group and the control group were cultured at 37 C, 5% C02 and saturated humidity incubator for 48 hours. Von Kossa staining kit was used to stain the OPLL cells in the experimental group and the control group. 9. Results: 1. The cytotoxicity of the co-culture system to the posterior longitudinal ligament cells was detected by cytotoxicity MTT assay. The results showed that the OD490 of the experimental group was higher than that of the control group, and the OD49o of the control group was 0.25 [0.06], and the OD49o of the experimental group was 0.28 [0.09], the difference was statistically significant (P 0.05). 2. The number of newly synthesized red cell nuclei with DNA in the experimental group was significantly higher than that in the control group. Within 4 hours, the proliferation rate of OPLL cells in the experimental group was 14.30 (+ 2.67%) and that of OPLL cells in the control group was 2.21 (+ 0.64%). The difference was statistically significant (P 0.05). 3. Von Kossa staining and ALP staining were detected in Von Kossa staining. The number of positive cells in the experimental group was significantly higher than that in the control group. 4. The expression of collagen type I, collagen type VII and osteocalcin mRNA in the experimental group was significantly higher than that in the control group. Compared with OPLL group and NP group, the levels of IL-1a, TNF-a and PGE2 in the supernatant of OPLL+NP group were significantly increased (P 0.05). The levels of IL-6 in the supernatant of each group were lower than the minimum detection standard of 6.25 pg/ml. Conclusion: In vitro, nucleus pulposus cells can be detected. The cervical disc herniation promotes the ossification of the posterior longitudinal ligament, and the cytokines secreted by the nucleus pulposus play an important role in the ossification of the posterior longitudinal ligament.
【學(xué)位授予單位】：山東大學(xué)
【學(xué)位級別】：碩士
【學(xué)位授予年份】：2016
【分類號】：R681.5
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