【作者】 熊晓芊；

【导师】 杨述华；

【作者基本信息】 华中科技大学 ， 外科学， 2006， 博士

【摘要】 一.烟酰胺对体外培养兔椎间盘基质的保护作用目的观察烟酰胺对椎间盘蛋白聚糖及Ⅱ型胶原的保护作用,并探讨其机制。方法构建兔椎间盘组织凝胶培养模型,以白介素-1β(IL-1β)诱发其退变。将其分为1~6组:第1组为正常对照,2~6组分别加入0.5mg/ml Nia;10ng/ml IL-1β;10ng/ml IL-1β及0.5mg Nia,10ng/ml IL-1β及0.25mg Nia;10ng/ml IL-1β及0.05mg Nia。培养至1周及2周时对各组标本藏红O-快绿染色,葡萄糖醛酸含量测定,以及aggrecan核心蛋白RT-PCR,培养至2周时行椎间盘Ⅱ型胶原RT-PCR及免疫组化检测,培养1周时行iNOS及TGF-β1免疫组化检测。结果①给予0.5mg/ml烟酰胺1周后髓核糖醛酸含量较正常对照组上升44.8%(P<0.01);给予IL-1β的各组糖醛酸含量随烟酰胺浓度上升而增高:0.5mg/ml治疗一周后髓核糖醛酸含量较未治疗组上升68.3%(P<0.01),两周后髓核及纤维糖醛酸含量仍高于同期正常对照组(P<0.01)和未治疗组(P<0.01)。②藏红O-快绿染色显示,随着烟酰胺浓度加大,髓核及纤维环染色浓度逐渐增大,且组织结构受IL-1β破坏逐渐减小。③RT-PCR显示给与IL-1β的各组椎间盘内,核心蛋白表达强度随烟酰胺浓度上升而上升。④Ⅱ型胶原免疫组化显示,治疗组板层结构完整性和胶原纹路连续性好于退变组。⑤RT-PCR显示治疗组Ⅱ型胶原表达明显强于退变组(P<0.01)。⑤第3组iNOS阳性染色率较第1组明显升高(χ2=148.374,P<0.001)。第4组阳性染色率较第3、5、6组均有明显下降(χ2=86.752, P<0.001;χ2=16.935,P<0.001;χ2=57.225,P<0.001)。⑥第3组TGF-β1阳性染色率较第1组明显下降(χ2=148.345,P<0.001)。第4组阳性染色率高于3、5、6组(χ2=56.686, P<0.001;χ2=7.685,P=0.008;χ2=39.143,P<0.001)。结论:体外条件下,烟酰胺可提高椎间盘聚集蛋白聚糖及Ⅱ型胶原含量,对抗IL-1β诱发的蛋白聚糖和Ⅱ型胶原含量下降。这种保护作用与其抑制iNOS的表达和保护TGF-β1的表达有关。烟酰胺具备用于椎间盘退变临床治疗的潜力。二.烟酰胺对体外培养兔椎间盘组织凋亡及能量代谢相关基因的影响目的观察烟酰胺(Nia)对白介素-1β(IL-1β)诱导的体外培养的椎间盘组织内细胞凋亡的抑制作用及能量代谢相关基因:HIF-1α(缺氧诱导因子-1α)、GLUT-1(葡萄糖转运蛋白-1)和VEGF(血管内皮生长因子)表达的影响。方法构建兔椎间盘组织凝胶培养模型,将其分为1~4组:第1组为正常对照,2~4组分别加入0.5mg/ml Nia;10ng/ml IL-1β;10ng/ml IL-1β及0.5mg Nia。培养一周后对各组标本行TUNEL、FAS、Caspase-3、Bcl-2、HIF-1α,GLUT-1及VEGF免疫组化染色。结果①TUNEL染色阳性细胞率分别为15.7%,17.0%,39.5%和31.3%,第4组较第3组有明显下降(χ2=1.555,P=0.212)。②Fas染色阳性细胞率分别为9.3%,8.5%,24.3%和22.8%,第4组与第3组接近(χ2=0.102,P=0.749)。③Bcl-2染色阳性细胞率分别为64.1%,73.7.%,49.3%和58.8.%,第4组较第3组有所增高,但并无统计学差异(χ2=2.832,P=0.092)。④Caspase-3染色阳性细胞率分别为3.4%、4.2%、17.6%和10.3%,第4组较第3组有明显下降(χ2=5.063,P=0.024)。⑤HIF-1α阳性细胞率分别为26.7%,4.2%,70.6%和45.3%,第4组较第3组有明显下降(χ2=29.792,P<0.001)。⑥GLUT-1阳性细胞率分别为25.7%,16.3%,56.3%和54.4%,第4组较第3组下降并不明显(χ2=0.135,P=0.713)。⑦VEGF阳性细胞率分别为10.1%,3.6%,68.4%和46.7%,第4组较第3组有明显下降(χ2=39.002,P<0.001)。结论烟酰胺可以抑制IL-1β诱导的椎间盘细胞凋亡。烟酰胺对椎间盘组织的能量代谢有促进作用,可以改善IL-1β导致的能量代谢障碍。三.烟酰胺对兔髓核细胞增殖及凋亡的调节作用目的考察烟酰胺(Nia)对髓核细胞增殖及凋亡的调控作用及其机制。方法体外培养兔髓核细胞,将其分为1~6组:第1组为正常对照组,不加入药物。2~6组分别加入0.5mg/ml烟酰胺,10ng/ml IL-1β,10ng/ml IL-1β及非特异性Caspase抑制剂Z-VAD-FMK,10ng/ml IL-1β及0.05mg/ml烟酰胺,10ng/ml IL-1β及0.5mg/ml烟酰胺。药物处理3天后对各组细胞行Annexin V-PI染色、Caspase-3、Caspase-8、Caspase-9功能的流式细胞仪检测,荧光显微镜照相及MTT法检测。结果①各组凋亡髓核细胞比例(Mean±SEM)分别为2.67±1.08%,2.71±0.53%,20.37±1.57%,11.34±0.67%,18.17±0.74%,9.42±1.08%。第2组较对照组无明显变化(P=0.950),第4、5、6组较第3组有所下降(P=0.001,P=0.172,P=0.001)。②Caspase-3功能阳性细胞比例由第3组的17.14±0.72%分别降至5、6组的12.35±0.64%(P<0.001)和9.26±0.36%(P<0.001)。③Caspase-9功能阳性细胞比例由第3组的19.4±0.98%分别降至5、6组的15.13±1.45%(P=0.014)和10.17±2.50%(P=0.004)。④加入烟酰胺后Caspase-8功能变化不大。⑤各组MTT检测测得的吸光度分别为0.972±0.064,1.023±0.086,0.519±0.021,0.637±0.028,0.956±0.013以及0.541±0.017,第6组吸光度远低于第3组(P<0.001),第5组吸光度与第3组接近(P=0.096)。结论烟酰胺可以促进髓核细胞的增殖和抑制IL-1β诱导的髓核细胞凋亡,对于凋亡的抑制作用主要通过抑制Caspase-9相关的线粒体凋亡途径实现的。四.可控压力致兔腰椎间盘退变模型的构建及其对纤维环能量代谢相关基因的影响目的构建“可控压力致兔腰椎间盘退变模型”,并考查该模型能量代谢相关基因:HIF-1α(缺氧诱导因子-1α)、GLUT-1(葡萄糖转运蛋白-1)和VEGF(血管内皮生长因子)的表达情况并探讨其意义。方法采用可控压力致兔腰椎间盘退变模型构建轻度兔椎间盘退变模型,Thompson分级及HE化染色观察退变程度。以10kg轴向压力获得加压24h组、72h组、加压24h后恢复48h组(恢复组)及正常对照组兔椎间盘标本,分离其纤维环组织,RT-PCR检测HIF-1α、GLUT-1基因的表达,Western Blot及免疫组化检测VEGF的含量及分布。结果①72h组椎间盘组织Thompson分级为Ⅰ级,HE染色显示纤维环纹路不及正常椎间盘清晰,部分细胞出现异常核型。②正常组仅见少量HIF-1α表达,24h组含量较正常组升高20余倍(t=25.022,P<0.001),72h组及恢复组较24h组依次下降。③正常组GLUT-1少量表达,24h组较正常组显著增高(t=18.314,P<0.001),72h组较24h组略有增强(t=2.819,P=0.023),恢复组与24h组接近。④正常组未检出VEGF,其余各组则均有明显表达。72h组免疫组化显示外层纤维环VEGF阳性细胞率高于内侧纤维环。结论可控压力致椎间盘退变模型可以有效地诱发压力导致的椎间盘退变。能量代谢相关基因表达的变化在压力致椎间盘组织损伤及损伤后修复的过程中扮演者重要角色。因此,将具有细胞能量代谢促进作用的烟酰胺应用于椎间盘退变的治疗和预防具有可靠的理论基础。五.烟酰胺治疗可控压力致兔腰椎间盘退变模型的实验研究目的考查烟酰胺对压力导致的兔椎间盘退变的治疗作用。方法构建可控压力致兔椎间盘退变模型。将24只日本大白兔随机分入1~6组:第1组2只,为假手术对照组;第2组2只,给与约50mg/kg烟酰胺口服1周;第3组5只,以10kg压力加压1周;第4组5只,以10kg压力加压1周,自行恢复1周,第5组5只,加压1周,给予约50mg/kg烟酰胺口服1周,第6组5只,给与约50mg/kg烟酰胺口服2周,这期间先加压1周,然后恢复1周。对各组标本进行形态学观察、HE染色、藏红O-快绿染色及Ⅱ型胶原免疫组化染色。结果①第2组椎间盘未见明显异常;第3组5只动物Thompson分级均为Ⅱ级,第4组4只为Ⅱ级,1只为Ⅲ级,第5组2只为Ⅰ级,3只为Ⅱ级,第6组3只为Ⅰ级,2只为Ⅱ级。②HE染色显示第4组椎间盘退变程度最严重,而第6组纤维环板层结构的连续性和完整性,髓核陷窝样结构的形态以及细胞的形态都较第4组有较明显的恢复。③第2组藏红O染色强度较第1组有所上升;第5、6组髓核和纤维环染色强度均高于第4组的相对应部位,其中以第6组髓核上升最为明显(29.9%和32.1%;P<0.01,P<0.01),第6组较第5组有轻微上升。④Ⅱ型胶原染色显示,第6组胶原的纹路连续性好于第4组;其中第6组外层纤维环染色强度较第4组外层纤维环升高约53.2%(P<0.01)最为明显,第6组各部位较第5组染色强度也有所上升。结论烟酰胺有助于减轻压力对椎间盘的损伤,能够促进压力损伤后的椎间盘恢复。更多还原

【Abstract】 1. Niacinamide protects rabbit intervertebral matrix in vitro.Objective To investigate the protective effects of Niacinamide on aggrecan and typeⅡcollagen of rabbit interverbral disc invitro. Methods Chiba’s 10ng/ml IL-1βinduced intervertebral disc (IVD) degeneration model was adopted in this study, and various concentration of Niacinamide was added to the medium for intervention: no Niacinamide as group 1, 0.5mg/ml Niacinamide without IL-1βas group 2, IL-1βwithout Niacinamide as group 3, IL-1βand 0.5mg/ml Niacinamide as group 4, IL-1βwith 0.25mg Niacinamide as group 5 and IL-1βwith 0.05mg/ml Niacinamide as group 6. At the 1st and 2nd week of culture, Safranin O- Fast Green Staining, glycosaminoglycan (GS) content measuring and aggrecan core protein RT-PCR was carried out to detect how aggrecan was regulated by niacinamide. At 2nd week of culture, RT-PCR and immunohistochemical staining was carried out for typeⅡcollagen. And after 1 week of culture, immunohistochemical staining for inducible nitric oxide synthase (iNOS) and transforming growth factor-β1 was carried out. Results①After 1 week’s culture, GS content of nucleus pulposus (NP) in group 2 and group 4 was raised 44.8% (P<0.01) and 68.3% (P<0.01) by 0.5mg/ml Nia as compared with group 1 and group 3 respectively. GS content of group 4, with 2 weeks of 0.5mg/ml Nia treatment, is higher than either group 3 (P<0.01) or group 1(P<0.01).②Safranin O- Fast Green Staining demonstrated that, with increasing of Nia concentration, staining density and histological structure of IVDs was better reversed.③RT-PCR showed that Nia increased core protein gene expression in normal IVDs, and up-regulated the expression in degenerated IVDs by a dose related mode.④Type Ⅱcollagen staining demonstrated that lamellar structure and continuity of collagen of treated groups was better reversed than the group3.⑤RT-PCR showed that expression of typeⅡcollagen of group 4 was significantly stronger than that of group 3 (P<0.01).⑥The rate of iNOS positive-staining cells of each group was 17.6%,10.9%,73.9%,19.3%,43.6% and 64.7% respectively. The positive rate of group 3 is siginificantly higher than group 3 (χ2=148.374,P<0.001) and the rate of group 4 is lower than group 3, 4 and 6 (χ2=86.752, P<0.001;χ2=16.935,P<0.001;χ2=57.225,P<0.001).⑦The rate of TGF-β1 positive-staining cells of group 3 is reduced as compared with group 1 (χ2=148.345,P<0.001). And the rate of group 4 is higher than either of group 3, 5 and 6 (χ2=56.686, P<0.001;χ2=7.685,P=0.008;χ2=39.143,P<0.001). Conclusion Niacinamide can raise content and expression of both aggrecan and typeⅡcollagen. And it is capable of inhibiting IL-1βinduced down-regulationg of aggrecan and typeⅡcollagen. The inhibition is related with its inhibition on iNOS expression and prection on TGF-β1 expression. Thus, Niacinamide offers a potential choice for IVD degeneration therapy.2. Niacinamide regulates cell apoptosis and energy metabolism related genes in cultured rabbit intervertebral discsObjective To investigate the regulatory effect of Niacinamide on IL-1βinduced cell apoptosis and energy metabolism related gene expression in intervertebral disc in vitro. Methods Chiba’s 10ng/ml IL-1βinduced intervertebral disc (IVD) degeneration model was adopted in this study, and various concentration of Niacinamide was added to the medium for intervention: no Niacinamide as group 1, 0.5mg/ml Niacinamide without IL-1βas group 2, IL-1βwithout Niacinamide as group 3, IL-1βand 0.5mg/ml Niacinamide as group 4. After 1 week of culture, TUNEL staining and immunohistochemical staining for FAS、Caspase-3、Bcl-2、HIF-1α,GLUT-1 and VEGF was used to detect alternated cell apoptosis and expression of energy metabolism related genes. Results①The rate of TUNEL positive-staining cells of each group was 15.7%,17.0%,39.5% and 31.3% respectively. The rate of group 4 is much lower than group 3 (χ2=1.555,P=0.212).②The rate of Fas positive-staining cells of each group was 9.3%,8.5%,24.3% and 22.8% respectively. The rate of group 4 is close to group 3 (χ2=0.102,P=0.749).③The rate of Bcl-2 positive-staining cells of each group was 64.1%,73.7.%,49.3% and 58.8% respectively. The rate of group 4 is higher than group 3, but not significantly (χ2=2.832,P=0.092).④The rate of Caspase-3 positive-staining cells of each group was 3.4%、4.2%、17.6% and 10.3% respectively. The rate of group 4 is significantly lower than group 3 (χ2=5.063,P=0.024).⑤The rate of HIF-1αpositive-staining cells of each group was 26.7%,4.2%,70.6% and 45.3% respectively. The rate of group 4 is significantly lower than group 3 (χ2=29.792,P<0.001).⑥The rate of GLUT-1 positive-staining cells of each group was 25.7%,16.3%,56.3% and 54.4% respectively. The rate of group 4 is significantly lower than group 3 (χ2=0.135 , P=0.713).⑦The rate of VEGF positive-staining cells of each group was 10.1%,3.6%,68.4% and 46.7% respectively. The rate of group 4 is significantly lower than group 3 (χ2=39.002,P<0.001). Conclusion Niacinamide can inhibite IL-1βinduced intervertebral disc cell apoptosis. Niacinamide improves energy metabolism of cultured IVD tissue and alleviates IL-1βinduced disturbance of energy metabolism.3. Niacinamide regulates rabbit nucleus pulposus cell apoptosis and proliferation in vitroObjective To investigate regulatory effects of Niacinamide on rabbit nucleus pulposus cell apoptosis and proliferation in vitro. Methods Cultured NP cells were divided 6 groups and various Niacinamide and IL-1βwas added to the medium: group 1 without any drugs as control, 0.5mg/ml Nia as group 2, 10ng/ml IL-1βand group 3, 10ng/ml IL-1βand non-specific Caspase inhibitor Z-VAD-FMK as group 4, 10ng/ml IL-1βand 0.05mg/ml Niacinamide as group 5, 10ng/ml IL-1βand 0.5mg/ml Niacinamide as group 6. After 3 days of co-culture with the drugs, the cells were examined with Annexin V-PI staining, Caspase-3、Caspase-8、Caspase-9 function staining and MTT assay. Results①The apoptotic rate (Mean±SEM) of the groups were 2.67±1.08%,2.71±0.53%,20.37±1.57%,11.34±0.67%,18.17±0.74% and 9.42±1.08%. The rate of group 2 did not change much than group 1 (P=0.950). The rate of group 4, 5 and 6 discended than group respectively (P=0.001,P=0.172,P=0.001).②The positive rate of Caspase-3 function staining of group 5 (12.35±0.64%) and 6 (12.35±0.64%) decreased as compared with group 3 (17.14±0.72%, P<0.001, P<0.001).③The positive rate of Caspase-9 function staining of group 5 (15.13±1.45%) and 6 (10.17±2.50%) decreased as compared with group 3 (19.4±0.98%, P=0.014, P=0.004).④The positive rate of Caspase-8 function staining did not change much with either Niacinamide or IL-1β.⑤The absorbance of the groups was 0.972±0.064,1.023±0.086,0.519±0.021,0.637±0.028,0.956±0.013 and 0.541±0.017 respectively. The A of group 6 is significantly higher than the A of group 4 (P<0.001), while the A of group is not significantly higher than group 3 (P=0.096). Conclusions Niacinamide is capable of impove cell proliferation and inhibite apoptosis of NP cells in vitro. The inhibition of apoptosis mainly acts via inhibition of mitochondrial path way.4. Estabilishment of Controlable Pressure Induced Rabbit Lumbar Intervertebral Disc Degeneration Model and Study of Alternated Expression of Energy Metabolism Genes of the ModelObjective To establish controlable pressure induced rabbit lumbar IVD degeneration model. And to investigate alternated expression of energy Metabolism genes of the model. Methods Twenty Japanese white rabbits were selected for animal model establishment. A“controllable pressure-induced rabbit IVD degeneration model”was adopted to impose various pressured on rabbit IVDs in vivo to obtain slightly degenerated rabbit IVD. Thompson’s gross grading system was used to access the degeneration degree of the IVDs and HE staining was used to check the histological changes followed with pressure damage. The survived animals whose IVDs were compressed successfully were divided into 4 groups randomly. The IVDs were treated with no pressure as control (group 1), with 10 kg axial load for 24 hours (group 2), 72 hours (group 3), and 24 hours with a 48 hours free for self-reparation (group 4). RT-PCR was used to detect the expression of HIF-1αand GLUT-1. Western Blot and immunohistochemical test were carried out for the content and distribution of VEGF. Results①After 72h’s over load, the IVDs of group 2 reached gradeⅠas assess ed by Thompson grading system. HE staining revealed that, the lamellar structure of AFs in group 2 was not as clear of that of normal control, and abnormal karyotype appeared more frequently.②HIF-1α: A very low expression was detected in normal AF, while the expression in group 2 was raised over 20 times as compared with normal (t=25.022, P<0.001). The expression in group 3 and group 4 decreased as compared with group 2.③GLUT-1 expressed weakly in normal AF. The expression in group 2 raised a lot as compared with control (t=18.314, P<0.001) and the expression in group 3 rose slightly than group 2 (t=2.819, P=0.023). The expression in group 4 is close to that in group 2.④Little VEGF content was detected in normal AF, while the content rose significantly in the other 3 groups. Immunohistochemical staining showed more VEGF positive stained cells in outer AF than in inner AF. Conclusion The“controlable pressure induced rabbit lumbar IVD degeneration model”is able to cause rabbit IVD degenerate effectively. Continuous pressure can strongly up-regulate expression of energy metabolism gene: HIF-1α, GLUT-1 and VEGF in vivo. These genes play important roles in AF adaptation and reparation in over load-caused damage. Thus, Niacinamide may be effective for IVD degeneration therapy.5. Niacinamide Treatment for Overload caused Rabbit Intervertebral Disc in vivoObjective To investigate therapeutic effects of Niacinamide for pressure caused rabbit intervertebral disc degeneration. Methods The“controllable pressure-induced rabbit IVD degeneration model”was adopted for estabilishing overload caused rabbit IVD degeneration model. Twenty four Japanese white rabbits were randomly divided into 6 groups with various pressure added to their lumbar IVDs, and with or without about 50mg/kg Niacinamide for oral administration per day: group 1, 2 rabbits, no pressure as fake operation control group; group 2, 2 rabbits, Niacinamide for 1 week without pressure; group 3, 5 rabbits, 10kg for 1 week without Niacinamide; group 4, 5 rabbits, 10kg for 1 week followed by 1 week’s free recovery; group 5, 5 rabbits, 10kg for 1 week followed by 1 week’s recovery with Niacinamide treatment and group 6, 5 rabbits, 10kg for 1 week with 1 week’s recovery, during which Niacinamide was given continuously. Thompson grading system was used to assess IVD degeneration degree, and HE staining was used to check the hitlogical changes of the samples. Safranin O- Fast Green staining and immunohistochemical staining for typeⅡcollagen was used to detect the alternated content and distribution of aggrecan and typeⅡcollagen. Results①There was no abnormal phenomenon in IVD samples in group 2 as compared with group 1. The 5 rabbits all grededⅡin group 3. There 4 rabbits gradedⅡand 1 rabbit gradedⅢin group 4. There 2 rabbits gradedⅠand 3 rabbits gradedⅡin group 5. And There 3 rabbits gradedⅠand 2 rabbits gradedⅡin group 6.②HE staining revealed that IVD of group 4 suffered the most severe degeneration. However, the continuity and integrality of lamellar structure of AF, the shape of lacune structure of NP and the appearance of cells in group 4 recovered obviously as compared with group 4.③Safranin O- Fast Green staining tensity of group 2 raised as compared with group 1. The staining tensity of group 5 and 6 rose as compared with group 4, among which the increase of NP was the most obvious (29.9% and 32.1%;P<0.01,P<0.01), and there was a slightly increase when comparing staining intensity of group 6 with group 5.④TypeⅡcollagen staining revealed that continuity of collagen lines of group 6 was better reversed as compared with group 4. The staining intensity of AF of group 6 increased 53.2% as compared with group 4 (P<0.01). Ane there was a slightly increase when comparing staining intensity of group 6 with group 5. Conclusion Niacinamide can help to allevate overload caused damage to IVD. And Niacinamide can benefit the recovery of damaged IVDs.更多还原