【作者】 李凯；

【导师】 杨述华； 卫小春；

【作者基本信息】 华中科技大学 ， 外科学， 2009， 博士

【摘要】 第一部分力学因素对体外培养兔软骨细胞糖胺多糖合成的影响实验一原代和传代兔关节软骨细胞糖胺多糖合成的动态观察目的研究传代对体外培养兔关节软骨细胞糖胺多糖(glycosaminoglycan，GAG)合成的影响。方法1月龄新西兰兔5只，无菌手术切取双膝关节软骨，采用0.4％Pronase酶和0.025％Ⅱ型胶原酶消化分离关节软骨细胞，体外培养。待细胞融合时，换无血清培养液。于换液后12、24、36、48、60h分别抽取上清液测量GAG浓度。传代两次，重复上述过程。采用重复测量资料的方差分析检验P0、P1、P2代细胞上清液液GAG浓度的差异。结果P2代以内，软骨细胞形态无明显变化，但P2代细胞内空泡状颗粒增多。传代后，细胞融合时间缩短，但是上清液GAG浓度随传代逐渐下降(P＜0.001)，且P0、P1、P2代之间两两比较差异有显著性(P＜0.001)。换液60h后，P1代软骨细胞GAG浓度较P0代下降24％，P2代较P0代下降74％。换液后时间越长，上清液GAG浓度越大(P＜0.001)。换液后时间与传代之间存在交互效应，时间越长，传代细胞与原代细胞上清液GAG浓度相差越大(P＜0.001)。结论在体外单层培养条件下，原代软骨细胞GAG合成能力最强，传代后很快大幅下降。细胞融合后连续检测上清液GAG浓度是研究软骨细胞分化的有效方法。实验二高密度细胞培养对兔关节软骨细胞糖胺多糖合成的作用目的观察不同接种密度下，软骨细胞合成糖胺多糖的能力。材料与方法1月龄新西兰兔5只。采用0.4％Pronase酶和0.025％Ⅱ型胶原酶消化分离双膝关节关节软骨细胞，来源于同一只兔的软骨细胞分为两部分，一部分以2×104／cm2接种，传代时仍以相同密度接种。另一部分在细胞贴壁后，人工降低细胞密度至2×103／cm2培养。倒置显微镜下观察细胞形态和增殖情况。原代和传1代细胞于细胞融合后换液。换液后12，24，36，48，60h以改良Alcian blue染色沉淀法测定糖胺多糖质量浓度。结果原代高密度培养组关节软骨细胞为多边形，轮廓清晰，三四天即可见集落形成，集落周边细胞较中心瘦长，为长多边形，传1代细胞形态无明显变化。低密度培养细胞早期散在分布，7d左右形成集落，细胞形态与高密度培养无明显差异。原代低密度培养软骨细胞长到融合所需时间较原代高密度培养细胞所需时间长。原代低密度培养组上清液中糖胺多糖质量浓度显著低于原代及传1代高密度培养组软骨细胞(P＜0.001，P＜0.05)，且时间越长，质量浓度相差越大。结论与低密度培养相比，平面高密度培养可提高软骨细胞合成糖胺多糖的能力，明显减缓软骨细胞的失分化速度，提示高密度培养更有利于软骨细胞维持表型，是软骨平面培养的较好方式。实验三不同强度循环拉伸对原代培养兔软骨细胞糖胺多糖合成的影响目的研究不同强度的循环动态拉伸(Cyclic tenisle strain，CTS)对原代培养的兔关节软骨细胞糖胺多糖(glycosaminoglycan，GAG)合成的影响。方法1月龄新西兰兔6只，无菌手术切取双膝关节软骨，采用0.4％Pronase酶和0.025％Ⅱ型胶原酶消化分离关节软骨细胞，来源于同一只兔的软骨细胞分为3部分，分别接种3块BioFlex培养板，通过Flexercell 4000系统分别给予正弦波形、0.3Hz、6h／d，强度分别为0％、5％、15％的CTS刺激。于加载后24、36、48、60h观察细胞形态，并分别抽取上清测量GAG浓度。采用重复测量资料的方差分析比较不同强度CTS刺激组上清液GAG浓度的差异。结果细胞力学加载后可见周边部软骨细胞由多角形变为纺锤形，沿培养皿半径垂直方向排列。随拉伸强度的增加，上清GAG浓度依次上升(P＜0.001)，且经两两比较有显著性差异(P＜0.05)。随时间增加，上清GAG浓度逐渐增加(P＜0.001)。时间与加载条件之间存在交互效应，时间越长，加载组与对照组上清GAG浓度相差越大(P＜0.001)结论CTS可促进单层贴壁关节软骨细胞合成GAG，作用效果随拉伸强度增大而增加。第二部分大鼠膝关节负重与非负重区软骨组织学对比分析目的观察大鼠膝关节软骨负重区与非负重区组织形态、基质蛋白多糖成分和Ⅱ型胶原分布差异。方法Wistar大鼠5只，切取双膝关节，固定，脱钙，包埋，沿矢状面整体切片，HE染色，PH值1.0和2.5阿尔新蓝染色，番红0染色，阿尔新蓝-番红0复染观察软骨形态结构和基质蛋白多糖成分，免疫组化检测Ⅱ型胶原分布并测量软骨厚度。分别观察负重区与非负重区软骨形态和基质染色差异，并利用图像分析系统，对基质成分染色深浅进行光密度定量，t检验统计分析。结果负重区与非负重区软骨厚度、细胞分布、形态结构均有较大差异。负重区软骨较非负重区明显增厚(P＜0.05)，软骨各层结构特征较非负重区更加明显。非钙化层负重区较非负重区阿尔新蓝浅染，而番红0深染，经图像灰度分析差异均有显著性(P＜0.05)，钙化层在上述二区染色无明显差异(P＞0.05)。复合染色负重区蓝染区域明显少于非负重区(P＜0.01)。负重区非负重区软骨非钙化层PH2.5阿尔新蓝染色明显较PH 1.0阿尔新蓝染色加深。免疫组化未发现负重区与非负重区Ⅱ型胶原分布存在差异(P＜0.05)。结论胫骨和股骨髁负重区较非负重区厚度增加，软骨各层结构特征更加明显。非负重区与负重区比较，软骨透明质酸含量明显增加，硫酸软骨素和硫酸角质素含量明显减少，而Ⅱ型胶原含量无差异。说明不同的受力环境造成软骨基质成分差异。同时提示临床用来修复负重区软骨缺损的非负重区软骨缺乏适应负重区力学环境的组织结构。第三部分运动对大鼠膝关节软骨的影响实验一大鼠跑步装置的设计与应用目的研制一种诱导大鼠进行定量匀速跑步运动的实验装置。方法以外购跑步机提供匀速运动平面，支架将自制罩盒架于跑步机上限制大鼠跑步范围。罩盒前部暗室诱导大鼠进入，中部强光刺激大鼠进入前部暗室，尾部与电击器连接的电极对大鼠进行电刺激驱赶。结果首次跑步，大鼠仅经过数次电击，即可在跑道内匀速跑步，间断电击可完成训练。3-4天之后，大鼠即可形成条件反射，只需很少的电击即可按设定速度跑步。实验中后期基本不需电击。结论此设计充分利用大鼠趋暗本能和学习能力，减少了电击刺激的影响，并且具有成本低，易于操控的特点是研究跑步运动对大鼠运动系统影响的有力工具。实验二长距离平板跑步运动对大鼠膝关节蛋白多糖分布的影响目的观察长距离平板跑步运动对大鼠关节软骨蛋白多糖分布的影响。方法Wistar大鼠10只，随机分为对照组和跑步组。对照组笼养，跑步组每天以20m／min速度，连续跑步1000m。连续训练45天后，处死大鼠，切取双膝关节，固定，脱钙，包埋，沿矢状面整体切片，HE染色，番红0染色。观察胫骨内侧平台负重区软骨形态结构和基质染色差异，利用图像分析系统，测量软骨厚度，并对软骨各层染色深浅进行光密度定量。结果跑步组软骨表面完整，非钙化层厚度较对照组显著下降(P＜0.05)。番红O染色，软骨负重区非钙化层平均光密度明显下降(P＜0.05)，其中表层下降66％(P＜0.001)，中层下降56％(P＜0.001)，深层与对照组无显著差异(P＞0.05)，而钙化层较对照组上升38％(P＜0.05)。结论连续长距离平板运动后，软骨表面完整，但是软骨厚度下降，蛋白多糖含量下降。提示连续长距离平板运动对软骨有一定的损伤作用。实验三长距离上坡跑步运动对大鼠膝关节软骨基质的损伤作用研究目的观察长距离上坡跑步运动对大鼠关节软损伤作用。方法成年雄性Wistar大鼠10只，随机分为对照组和跑步运动组，每组5只，对照组笼养，跑步运动组每天以20m／min速度，连续水平跑步1000m，连续训练15d后，改为20°上坡跑，速度距离同前，上坡训练30d后，处死大鼠，切取双膝关节，固定，脱钙，包埋，沿矢状面整体切片，HE染色，番红0染色观察软骨形态结构和基质染色差异，采用OARSI评分对胫骨平台和股骨髁软骨损伤进行评价。结果上坡运动组软骨表面印度墨水染色显示表面粗糙，切片显示软骨出现明显的损伤，股骨髁与胫骨平台软骨OARSI评分明显高于对照组(P＜0.01)。损伤修复组织Ⅱ型胶原免疫组化阴性。结论连续长距离上坡运动，可造成软骨应力负荷增加，导致软骨发生明显损伤。第四部分膝骨关节炎放射学骨赘表现与内翻角相关性分析目的探讨膝骨关节炎患者放射检查骨赘表现与膝关节内翻角的关系。方法利用自制X光测量板对膝骨关节炎患者13例，共18膝，行DR照相，测量膝关节内翻角。利用Photoshop软件通过数码影像测量骨赘长度。骨赘长度与胫骨平台宽度的比值，称为突起指数，用以排除个体大小差异和投照放大等因素对骨赘长度测量的影响。应用SPSS软件通过Pearson相关检验探索膝关节骨赘大小和外翻角之间的关系。结果内翻角与股骨内侧平台骨赘长度有正相关性(r_s=0.585，P=0.014)，与胫骨内侧骨赘突起指数有较高的正相关性(r_s=0.761，P＜0.001)，内翻角与膝关节外侧室骨赘突起指数间尚不能认为有相关性存在。结论膝骨关节炎患者内侧室骨赘的大小与膝关节内翻程度有关，软骨异常受力后分泌细胞因子变化可能是此现象的原因。提示软骨细胞力学生物学研究对阐明骨关节炎的发病机理很有价值，有待进一步研究。更多还原

【Abstract】 PARTⅠ: Effect of Mechanical Strain on Cultured Rabbit ArticularChondrocyteExperiment 1: Effects of passage on the glycosaminoglycan synthesis of the culturedrabbit articular chondrocytesObjective: To study the effect of passage on the GAG synthesis of cultured rabbit articularchondrocytes.Methods: Chondrocytes were isolated from the knee joints of five one-month rabbit knees.The morphology of the cells was detected under inverted microscope. The GAGconcentrations of supematants were measured by precipitation with alcian blue at 24, 36,48, 60h after the confluent of the Passage 0,1,2 cells.Results: The GAG concentrations of the supernatants increased with time （P＜0.001）. Itdecreased with the increase of passage （P＜0.001）. And post hoc tests showed the significantdifference between any two passages （P＜0.05）.Conclusion: The GAG synthesis of in vitro cultured chondrocytes decrease with thepassages.Experiment 2: Effect of high-density cell culture on the synthesis ofglycosaminoglycan in rabbit articular chondrocytesObjective: To observe the performance of chondrocytes synthetizing GAG at differentinoculum densities.Methods: Five New Zealand rabbits of one month old were used in this study. Articularchondrocytes were isolated from both knees and digested using 0.4％pronase enzyme and0.025％Ⅱtype collagenase. The chondrocytes harvested from the same rabbit were dividedinto two sets, one was seeded at a constant density of 2×10⁴/cm₂ in primary and subculture, the other was cultured at a reduced density of 2×10³/cm² following cellular adhesion.Cellular morphology and proliferation were observed under inverted microscope. Theculture media were renewed after the primary cells and passage 1 cells were confluent.GAG concentration was determined using the modified precipitation method with Alcianblue at 12, 24, 36, 48 and 60 hours following the renewal of culture media.Results: Articular chondrocytes in the primary high-density culture group were polygonalwith clear boundaries, they have shown to form colony at 3-4 days. Cells around colonieswere more slender than those in the center of colonies, shaping as long polygon. There wasno obvious change observed in the morphology of passage 1 cells. In the low-densityculture group, cells scattered at early stage and formed colonies at 7 days, cellularmorphology showed no significant differences in comparison with high-density culturegroup. The time of primary cells becoming confluent in the low-density culture group wasprolonged compared with high-density culture group. The GAG concentration insupernatants in the primary cells of low-density culture group was significantly lower thanthat in primary cells and passage 1 cells of high-density culture group （P＜0.001, P＜0.05）.The GAG concentration showed a greater difference along with the prolonging of culturetime.Conclusion: High-density culture is better then low-density culture to enhance theperformance of chondrocytes synthetizing GAG and to retard the velocity of chondrocytesdedifferentiation, which suggests high-density culture contributes to maintain thechondrocytes phenotype and can be considered as a good way of plate culture.Experiment 3: Effects of cyclic mechanical strain with varying amplication on theglycosaminoglycan synthesis of the primary cultured rabbit articular chondrocytesObjective: To study the effect of cyclic tensile strain （CTS） with varyingamplication on the GAG synthesis of primary cultured rabbit articular chondrocytes. Methods: Cyclic tensile strain （sinusoidal wave, 0.3Hz, 6h/d）of different amplication （0％,5％, 15％） was applied to monolayer cultured primary rabbit articular chondrocytes using aFlexercell 4000 strain unit. The morphology of the cells was detected under invertedmicroscope. The GAG concentrations of supernatants were measured by precipitation withalcian blue at 24, 36, 48, 60h after the beginning of the first CTS loading.Results: The cells in the middle of dishes exhibited morphologic change from a polygonalto spindle-like shape and lined perpendicularly to the radius of the dishes. The GAGconcentrations of the supernatants increase with the CTS amplication （P＜0.05）.Conclusion: CTS enhance the GAG synthesis of cultured primary rabbit articularchondrocyte with an amplication-dependent manner.PartⅡ: The Histological Compare of Articular Cartilage of the Loadingand Unloading Area in Rat Knee Joints.Objective: To observe and analysis the morphology and matrix content in articularcartilage of the loading and unloading area in rat knee joints. Methods: Wistar Knees of 5wistar rats were studied by whole-mount section technique. The sections were stained withhaematoxylin, eosin, alcian blue and safranin O to elucidate the morphological differenceof loading and unloading area. The cartilage thickness and stain difference were compared.Results: There is great different in the loading and unloading area in the chondrocyte arrayand cartilage thickness. The cartilage of the unloading area has less chondroitin sulfate（CS）and keratan sulfate（KS） but more hyaluronic acid （HA）.Conclusion: The unloading area of the articular cartilage is different from loading area histologically. It elucidated that the graft of unloading area has no structure suitable to themechanic environment of the loading area in mosaicplasty.PartⅢ: The Effect of Sports on Articular Cartilage of RatExperiemnt 1: The Design and Application of Rat TreadmillObject: To develop a rat treadmill.Method: The treadmill machine has been designed to compulsively make animals exerciseby manual controlled electrical shock. The shock is delivered to the animals by theelectrode at the end of the machine. The fore part of the treadmill was dark and the middleof the treadmill was strongly lighted. The rats would see the strong light before beingshocked. After 45 days intensive running exercise （1km/d、20m/min）, rat knee cartilagestudied by whole-mount section and safranin O stain technique and compared with control.Result: The rats soon learned to run back to the dark part of the treadmill after severaltimes of shock stimulation. And in 2-3 days, they can learn to run in the treadmill withoutshock stimulation. The safranin O staining of uncalcified cartilage was reduced in therunning rats group （P＜0.05）.Conclusion: These designs make full use of the instinct to dark place and learning abilityof the rats, so it greatly reduced the shock. The it provide a good tools to study the effect ofsports on muscle-skeletal system. Experiment 2: Articular Cartilage Glycosaminoglycan Distribution in the rat KneeJoint After Strenuous Running Exercise.Object: To study the influences of the strenuous running training program on the knee jointarticular cartilage.Method: At the age of 16 weeks, 5 male wistar rats started running on a horizontaltreadmill. Thereafter, the rats were trained for 45 days continuously. The rats ran 1km/d atthe speed of 1.2km/h. 5 matched male rats served as controls. Knees of the rats werestudied by whole-mounted section, HE stain, safranin O stain.Result: The cartilage surfaces were intact after the running exercise. Theglycosaminoglycan concentration was decreased an average of 66％in the superficialzone, 56％on the middle zone, but no difference was found in the deep zone. And theglycosaminoglycan concentration was increased 38％in the calcified cartilage.Conclusion: Strenuous running induced marked decrease of proteoglycan in theuncalcified cartilage, but induced no osteoarthritis in this rat strenuous running model.Experiment 3: The Effect of Strenuous Uphill Running on the Articular Cartilage ofthe KneeObject: To study the influences of the strenuous uphill running training program on thearticular cartilage of the knee.Method: At the age of 16 weeks, 5 male wistar rats started running on a nearly horizontaltreadmill. Thereafter, the rats were trained for 45 days continuously. The rats ran 1km/d atthe speed of 1.2km/h in the first 15 days and 20°uphill in the last 30 days. 5 matched malerats served as controls. The knees of the rats were studied by whole-mounted section, HEstain, safranin O stain, immunochemistry staining for colleganⅡand OARSI score.Result: The cartilage surfaces strained with india ink became irregular and OARSI scoresignificantly wordened after 45 days training. Conclusion: Strenuous uphill running induced osteoarthritis in the knee joints of rats. Theincreased loading of the cartilage may be the reason.PARTⅣ: Correlation between radiographically diagnosed osteophytes and varusangle in the knees of osteoarthritis patients.Objective: To assess the correlation between the presence of radiographically diagnosedosteophytes and varus angle in the knees of osteoarthritis patients.Methods: A total of 18 knees in 13 patients with knee OA was studied. The length ofmarginal osteophytes that appeared in radiogram was measured. A special scale methodcalled the "spur index" was devised for the study to determine a corrected measurement ofthe osteophyte length, varus angle were measured with Limb X Film Measuring Plate（LXMP）. The correlation between varus angle and osteophytes formation was evaluated byPearson correlation.Results: No correlation between the osteophytes of lateral tibiofemoral compartments andvarus angle was found. In medial tibiofemoral compartment the osteophytes’ lengthcorrelates positively with varus angle （P＜0.05）.Conclusion: This study demonstrate a strong positive association between the varus angleand osteophytes’ size in medial tibiofemoral compartment. The secretion of cytokines afterabnormal mechanical stimulation may be the reason of this finding. More studies about themechanobiology of chondrocyte will be needed to confirm this finding and test ourhypothesis.更多还原