| Osteoarthritis (OA) is a common, multiple degenerative disease, however the cause isnot clear so far. It is considered by a complex interactions of the genetic, metabolism,biochemical and biomechanical factors, activation of inflammatory reactions, includingthe action of cartilage, subchondral bone and synovial membrane, and thus induce thearticular cartilage injured. It’s mainly concernd with the chondrocyte nutrient metabolism,abnormal degradation of extracellular matrix (ECM) and biomechanical balance.Chondrocytes are exposed to a variety of biological and mechanical signal afterconduction pressure of the cartilage ECM. OA is a multifactorial disease. Processesresulting in dissolution of articular cartilage are more complicated than expected, so that itis very difficult to analyse a factor alone. However, we ensure the loading ofbiomechanical is essential for the maintenance of cartilage’s homeostasis. Stimuli of thephysiological biological stress plays a key role in the growth and development of cartilagearticularis. Interaction of mechanical factors and the regulatory mechanisms ofmechanical factors between cells and tissues in both normal and pathological conditionsare not yet entirely clear. ILs and MMPs family are important inflammatory factor involving the pathogenesis of osteoarthritis. Chondrocytes regulate secretion of collagen,proteoglycans and inflammatory cytokines by combining mechanical signal to it’s geneexpression.MiRNA are small non-coding, single-stranded RNA molecules of a size of about22bases, which negatively regulate gene expression by regulating the expressioncomplementary target mRNA and affecting its translation into proteins. MiRNA playscrucial roles in regulation to all cells during process of cartilage’s formation andremodeling. Abnormal miRNA expression profiling has been proved to be closelyassociated with the development of OA.Normal and OA population for the study, simulated body environment, builded cyclichydrostatic pressure model. Researched loading the different intensity, different timeperiodic hydrostatic pressure leaded to effect of the normal and OA human kneechondrocytes biology, comparative differences.Objective: To improve the efficiency and success rate of chondrocytes cultured in vitroand build tissue cartilage engineering, To explore the appropriate time parameters ofpressures stimulate in the certain physiological range, for providing a theoretical basis forthe experiment. While exploring the pathogenesis of OA biomechanical factors lay someexperimental basis.Methods:1: Primary chondrocytes cultured in vitro; toluidine blue and type II collagenimmunocytochemical staining were applied to identify; MTT analysised chondrocytegrowth curve; establishing hydrostatic loading model.2: Normal chondrocytes were divided into four groups,2MPa loaded0h,4h,8h,12h,type II collagen immunocytochemical staining were used for the semiquantitative analysis;MTT assay analyzed information of chondrocytes cell multiplication in each group. Flowcytometer measured the apoptosis rates.3:(1) Normal chondrocytes were divided into pressure group loaded by10MPa for2h and the control group, OA chondrocytes was OA group. Detecting the content of IL-1β,MMP-3and GAG after loaded for5days.(2) Normal chondrocytes were divided into four groups,2MPa respectively pressurized0h,4h,8h,12h. after5days, detected the contentof IL-1β, MMP-3and GAG.4:(1) Real-time quantitative PCR detected the mir-21expression levels ofchondrocytes in10MPa pressure group,2MPa4h group,8h group,12h group and acontrol group.(2) After transfected has-mir-21mimics, observing the proliferation,apoptosis and expression of type II collagen of chondrocytes.Results:1: chondrocyte growth curve was shaped as "S", a slower proliferation during1-3day, proliferation significantly during5-8day, exponentially. At the8day reached a plateau,and tended to decline.2: With2MPa hydrostatic pressure, type II collagen content of cartilage cells in eachpressure group increased, cell proliferation accelerated, apoptosis rate decreased,especially8h group was the most significant.3:(1) There is a positive correlation between IL-1β and MMP-3levels in10MPapressure group, control group and OA group. IL-1β, MMP-3levels of cells in OA groupwere higher than pressure group and control group, IL-1β and MMP-3levels in pressuregroup were higher than control group. GAG concentration of pressure group decreasedsignificantly, and more significantly in early.(2)2MPa pressure grouped by time: controlgroup IL-1β levels (5.06±0.08pg/ml), MMP-3levels (5.33±0.74ng/ml) were the most,8hgroup IL-1β levels (3.79±0.32pg/ml), MMP-3levels (2.89±0.37ng/ml) were theminimum.8h group GAG content (452.80±19.96ug/ml) was were the most, control groupGAG content (245.70±17.57ug/ml) was the minimum.4:(1) Real-time quantitative PCR analysis, there were significant differences inmir-21expression levels of the six groups cells. OA group was highest,10MPa groupfollowed, three time groups with2MPa were less than control group.(2) After transfectionmir-21mimics, the chondrocyte proliferation rate was inhibited and decreased, theapoptosis rate of chondrocytes (23.63±3.38%) was increased, the expression of type IIcollagen was decreased.Conclusion: Chondrocytes loaded under hydrostatic pressure model in vitro wassuccessfully constructed. Hydrostatic pressure with2MPa in8h was conducive to enhance chondrocyte proliferation activity, reduced the apoptosis rate, promote the secretion ofCOL-II and GAG. The expression of mir-21significantly increased in OA chondrocytesand under10MPa hydrostatic pressure in2h.2MPa pressure stimulation during4-8h caninhibit the expression of mir-21. Upregulating the expression of mir-21can affect theactivity of chondrocytes. |
PDF Full Text Request