| BackgroundKee Osteoarthritis(KOA)is a kind of chronic degenerative diseases.OA is a major public health problem in the elderly population.The incidence rate of OA was approximately 8.1%in middle and elderly population in China.Incidence of OA among women was far beyond that among men.It is more common in rural areas than in urban areas.KOA is mainly correlated with age and obesity,which is mainly caused by spontaneous inflammatory reaction.The main method for the clincal treatment of OA include drug therapy,physical therapy and surgical therapy at present.While the curative effect is poor.Therefore,it is still need to develop new treatments.In recent years,research on tissue engineering and regenerative medicine has provided new ideas for the treatment of KOA.Treatment of OA by the precartilaginous stem cells(PSCs)have also provided theoretical basis for the application in KOA.Based on this,animal experimental research on the application of PSCs in KOA was carried out in the present study.Objective:By establishing the rat KOA model,animal experiments were conducted applying PSCs cell therapy for KOA treatment.The effect of PSCs therapy on the animals was analyzed by analyzing the expression of Tumor necrosis factor-a(TNF-α),Interleukin-1β(IL-1Β)and IL-6 at mRNA and protein levels.The morphology of cartilage of the knee joint was observed under the light microscope.We further analyzed the therapeutic effect of PSCs on tissue injury.In addition,the Notchl signaling pathway mediating the inflammatory response of KOA was explored to investigate the mechanism of PSCs on KOA.In addition,we explored the mechanism of PSCs alleviating joint injury by determining the expression of apoptotic factors(Bcl-2 and Bax).In conclusion,we aimed to provide experimental evidences in vivo for the application of PSCs therapy.MethodsChapter 2:The effect of PSCs treatment on cytokineClassic Hulth method was adopted to establish the rat model of KOA.The right hind limb knee hair of rat was removed and disinfected after anesthetization.We longitudinally cut the medial parapatellar skin of the right knee joint and the medial collateral ligament.Then the joint cavity was opened until the patella was completelyexposed.Afterthat,we bent right knee joint,cut the anterior cruciate ligament,removed the meniscus,stopped bleeding and cleaned the joint cavity.Finally,we put the patella back in its original position and seal it.Rats were sacrificed by cervical dislocation referring to Robinson’s method.La Croxi ring was taken from the femur and tibia epiphysis of rats.We centrifuged and collected the cells after digestion by 25%trypsin and type I collagenase.Thereafter,we incubated the cells at 37℃ in an incubator containing 95%of air and 5%CO2,for 1-2 days.When the cell fusion degree reached 80%-90%confluence,25%trypsin was used to digest the cells.The PSCs were then purified by immunomagnetic beads sorting system.The cells were washed rapidly,and the eluted cells which were positive cells were purified PSCs.The second generation cells were then cultured.Cultured cells at 3rd passage were applicable to injection into the articular cavity of rats.SD rats were randomly divided into 3 groups(N = 20 in each group):Control group,Model group and Treatment group.The rats in the Control group were did not performed the Hulth surgery and were directly injected with 100 μL phosphate buffer solution(PBS)into articular cavity after 3 days’ growth.As for the Model group,the rats were injected with 100 μL PBS into articular cavity on the third day after surgery.For the Treatment group,the rats were injected with 5 x 106 PSCs into the articular cavity on the third day.Blood and articular cartilage tissue samples were collected after 4 weeks and 8 weeks.Rats were anesthetized with chloral hydrate in the abdomen.Blood was drawn from rats by heart puncture.The supernatant was centrifuged at room temperature for 2 h.The expression of inflammatory factors(TNF-α,IL-1β and IL-6)in serum of rats were determined byELISA.After anesthesia,the rats were killed by dislocation of the neck.The cartilageof the medial femur was cut off in order to subsequently observe the tissue morphology.And the Western blot was performed to assess the expression of Notch 1,Bcl-2 and Bax at protein level.Chapter 3:The effect of PSCs on the histological morphology of rat KOA cartilageCartilage was fixed with formaldehyde and decalcified for 4 weeks.And then,it was embedded with paraffin.The tissue was cut into slices(4 μm)with a paraffin slicer.Whereafter,we unfolded the slices in water at 40℃ and removed the slices with a glass slide coated with 0.2%chromium potassium sulfate dodecahydrate.Then,the paraffin slices were placed on the dryer and baked for 2 h and dewaxed.Different concentrations of alcohol were used for hydration.Thereafter,the paraffin slices were stained with hematoxylin for 20 min.The differentiation was examined by rapid microscopy with 1%HCl alcohol.The paraffin slices were dehydrated with alcohol after rinsing with distilled water.And then,the slices were stainned with HE for 90 s and treated with xylene for 10 min after dehydrated with alcohol.The film was sealed with neutral gum finally.The sealing film was placed under 200 x microscope of Olympus optical microscope.Then,we observed the articular cartilage tissue injury degree of mices in the control group,model group and treatment group at 4 weeks and 8 weeks respectivelyChapter 4:PSCs therapy inhibits the Notchl signaling pathway and regulates the balance between Bcl-2 and Bax expression to alleviate knee cartilage injuryWestern blot was used to detect the protein expression levels of Notchl,Bcl-2 and Bax of mices in the KOA model group,PSCs treatment group,and control group.The protein was collected after RIPA lysis.Proteins were subjected to SDS-PAGE separation.We obtained the target protein after transmembrane.After the immunoreaction with the primary antibody and the secondary antibody marked by HRP,The membrane carrying blots and antibodies were covered with immobilon Western chemiluminescent HRP substrate.Protein signals were detected and quantified.β-actin were used as an internal controlResults:Chapter 2:The effect of PSCs therapy on inflammatory factors.KOA rat model was constructed by partially removing meniscus,and then the PSCs were directly injected into the keen joint.ELISA experimental results showed that the concentration of IL-1β was 32.42士 4.12 pg/mL in the serum of KOA model rats after four weeks,while it was 12.09士 2.31 pg/mL in the serum of the control rats which was nearly three times less than that in the KOA model.The concentration of IL-1βwas 22.43± 3.21 pg/mL in the serum of the treatment rats after four weeks which was significantly lower than model rats’(P<0.05).The concentration of IL-1β was 38.97± 4.52 pg/mL in the serum of KOA model rats after eight weeks,and it was obviously higher than the corresponding index four weeks ago(P<0.05).The concentration of IL-1β was 17.21 ± 2.67 pg/mL in the serum of the treatment rats after eight weeks,and it resulted in a 2.5 times reduction compared to the model rats.The concentration of TNF-a was 25.35± 4.31 pg/mL in the serum of KOA model rat after four weeks,it was nearly 2.5 times higher than Control rats’.The concentration of TNF-a was 19.31± 3.45 pg/mL in the serum of the treatment rats after four weeks which was significantly lower than model rats’(P<0.05).The concentration of TNF-a was 32.34 ± 3.12 pg/mL in the serum of KOA model rats after eight weeks,and it was obviously higher than that four weeks ago(P<0.05).Compared to control rats,the concentration of TNF-a was significantly increased(P<0.05).The concentration of TNF-a in the serum of the treatment rats was 15.31 ± 2.13 pg/mL which reduced 2 times compared with model rats.The concentration of IL-6 was 34.04± 5.65 pg/mL in the serum of KOA model rats after four weeks;it was nearly 4.25 times higher than Control rats’(8.03 士 2.15 pg/mL).The concentration of IL-6 was 46.23 士 5.23 pg/mL in the serum of the KOA model rats after eight weeks which was nearly five times higher than control rats’(9.76 ± 2.34 pg/mL).The concentration of IL-6 was 25.45 ±4.67 pg/mL and 20.78 ± 2.16 pg/mL in the serum of the treatment rats after four weeks and eight weeks which were significantly lower than the model rats’.The concentration of IL-6 in the serum of the treatment rats reduced nearly 2.5 times compared with the model rats’Chapter 3:Histological morphology of rat KOA cartilage’s change after PSCs therapyThe cartilage surface of control rats was smooth and chondrocytes was evenly distributed,arranged in neat rows,and had complete tidal line after four weeks.While the cartilage surface of model rats showed sharp crack and shallow ulcer,and the cartilage surface showed fibrosis.Furthermore,extensive osteophyte and incompleteness tidal line developed in the model rats.After PSCs treatment,the cartilage injury and osteophyte formation were significantly alleviated,and the tide line and the cell layer became clearer.Although the synovial membrane remainddamaged,the chondrocytes arranged evenly.No significant changes were observed in the control group after eight weeks.Cartilage damage in the model group was more obvious than that in the KOA Model rats after four weeks which showed defect on the cartilage surface,disordered arrangement of cartilage cells,distinct loss of cartilage,unclear cell layer,and disappearance of tidal line.The cartilage morphology of treatment rats treated with PSCs significantly improved the smooth surface of cartilage,and the cartilage had orderly arrangement of cartilage cells,clear cellular layer and complete tidal line.The improvement of cartilage morphology in the treatment group treated with PSCs was significantly better than that in the treatment group after four weeks.Chapter 4:PSCs therapy inhibits the Notchl signaling pathway and regulates the balance between Bcl-2 and Bax expression to alleviate knee cartilage injuryThe expression of the Notchl in the KOA model rats was significantly higher than the control rats after four weeks(P<0.05),and it was more significantly increased in the KOA model rats after eight weeks(P<0.05).After four weeks treatment,the expression of Notchl was significantly reduced in the treatment rats compared with model rats,and we could find the same results after eight weeks(P<0.05).The expression of Bcl-2 and Bax in the KOA model rats and PSCs treatment rats was all significantly higher than the control rats’(P<0.05).The ratio of Bcl-2 to Bax in the KOA model rats was significantly lower than control rats’(P<0.05).The ratio of Bcl-2 to Bax in the treatment group rats was significantly higher than the KOA model rats’ and control rats’(P<0.05).The same results were found after eight weeks treatment.Conclusions:Experiments in vivo showed that PSCs cell therapy could alliviate KOA injury.The expression of inflammatory cytokines(TNF-α,IL-1β and IL-6)was measured by ELISA.In addition,the therapeutic effect of PSCs cell therapy on cartilage injury of knee was further observed.Moreover,the regulatory effect of PSCs on Notchl signal pathway and its mechanism were further analyzed.Based on this study,we concluded:1.PSCs treatment could inhibit the secretion of cytoinflammatory(TNF-α,IL-1β,and IL-6)expression;2.PSCs treatment could alleviate the cartilage injury of KOA.With the increasing of the treatment time,the therapeutic effect of PSCs on the cartilage injury of KOA is more obvious;3.PSCs treatment could inhibit the activation of Notchl signal pathway,and PSCs may alleviate the knee cartilage injury by blocking the Notchl signal pathway;4.PSCs may alleviate knee injuries by maintaining expression balance between Bcl-2 and Bax. |
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