Objective To investigate the role of KAT7 in cartilage cell and tissue ageing by establishing an over-replicating-induced primary mouse cartilage cell ageing model and a mouse natural ageing model.Methods Chon-drocytes of the mouse knee joint were obtained by type Ⅱ collagenase digestion and identified by toluidine blue stai-ning and Col Ⅱ staining.The age-related proteins and KAT7 expression levels in cartilage cells from different gener-ations of mice were discovered using Western blot and cellular immunofluorescence techniques,and the aging of the cells was assessed by SA-β-Gal coloring.The pathological alterations were examined in the joints of 22-month-old mice compared to 2-month-old mice using HE staining and safranin O-solid green staining.Additionally,immuno-histochemical analysis was done to observe the expression of KAT7 and p53 in mouse joint tissue.Results Com-pared with the control group,the expression levels of KAT7 protein and p21 and p53 in aged mouse chondrocytes significantly increased.WM-3835,a commonly inhibitor of KAT7 that possess the capacity on halting the protein expression procedure of gene KAT7 as well as p21 in ageing chondrocytes.SA-β-Gal staining showed a significant increase in positive staining of chondrocytes in the eighth generation(P8)compared to the first generation(P1).Compared with the cartilage tissue of young mice,the cartilage tissue of elderly mice presents a near-bone distribu-tion,with a decrease in cartilage surface integrity,a significant increase in the number of hypertrophic chondro-cytes,and more KAT7 and p53 cells that were positive.Conclusion The expression of KAT7 increases in the ageing chondrocytes and the cartilage tissue of ageing mice,reveales the potential significance of KAT7 correlated to cellular aging process in cartilage.

Objective To examine the impact and partial mechanism of bupivacaine sustained-release drug(code PB21)in combination with low-dose dexamethasone(Dex)on the analgesic time of rats with knee osteoarthritis(KOA).Methods Using the techniques of anterior cruciate ligament transection and meniscus instability,a rat KOA model was created.After eight weeks,SD mice were split into three groups at random:a group for the model,one for Dex(50 μg),one for PB21(1.5 mg),and one for combined administration(1.5 mg PB21/50 μg Dex),with a control group that received a sham operation.The pain thresholds of KOA rats were measured using a Pres-sure Application Measurement(PAM)at different intervals before to delivery and 4,24,36,and 48 hours following administration;to gauge changes in discomfort,a CatWalk was used to assess the rats'average foot strength and maximum contact area before,four,twenty-four,and forty-eight hours after treatment.A portion of the rats were put to sleep at four,twenty-four,and forty-eight hours following the injection,and the joint synovium was removed for paraffin sectioning.Immunohistochemistry was used to identify the expression of GAP43 in the synovium,whereas immunofluorescence was used to identify the expression of CGRP in the same tissue.Results The average strength and maximum contact area of the foot and claw decreased(P＜0.01),and the pain threshold decreased(P＜0.01)in the model group compared to the sham operation group.The PB21+Dex group experienced a delayed pain threshold lowering time delay when compared to the PB21 and Dex treatment groups alone.Up to 48 hours lat-er,the combination administration group's average strength and maximum contact area of the foot paw remained ele-vated,and there was a statistically significant difference(P＜0.05)between the combined administration group and PB21 and Dex alone.GAP43 and CGRP expression levels in synovial tissue were detected.The results indica-ted that PB21 and Dex alone could lower protein expression levels at 4 and 24 h at the two time points,and that the PB21+Dex group could still significantly lower GAP43 and CGRP expression levels at 48 h.At the 48 h time point,the PB21+Dex group was statistically significant when compared to the PB21 and Dex alone administration group(P＜0.05).Conclusion In summary low dose dexamethasone can prolong the analgesic effect of PB21 on KOA rats,which is connected to reducing the expression of pain related proteins CGRP and GAP43.

Objective : To investigate the partial mechanism and effect of transplanting with human umbilical cordderived mesenchymal stem cells (hUC⁃MSCs) to repair articular cartilage defects in a rat model. Methods : Critical⁃sized osteochondral defects were created in the trochlear grooves of rat femurs. The hUC⁃MSCs were transplanted into the defect of experimental knees. Sham group and model group knees were transplanted with saline. The effects of articular cartilage repair were evaluated at 10 weeks after surgery with International Cartilage Repair Society (ICRS) repair score , histological examination and immunohistochemical analysis. The effects of hUC⁃MSCs on the proliferation and migration of chondrocytes were detected by Transwell in vitro. Results : After transplanting with hUC⁃MSCs , the articular surfaces of the defect sites were changed smoother thanthose of the model group , and the cellular morphology and arrangement were also improved in Safranin O staining or Masson staining results. Similar to surrounding normal articular cartilage tissue after treatment for 10 weeks , and the ICRS repair score was signifision of type Ⅱ (Col Ⅱ ) collagen and decrease the level of type Ⅰ collagen( Col Ⅰ ) in the articular defect site. Meanwhile , the increased protein expression of SOX9 and protein transportation to the nucleus were observed after treatment with hUC⁃MSCs for 10 weeks. In vitro , chondrocytes could be proliferated and migrated after co⁃cultured with hUC⁃MSCs. Conclusion Transplanting with hUC⁃MSCs can promote cartilage repair, and its role maybe related to the protection of the cartilage matrix and the promotion of chondrocyte proliferation and migration.

Objective : To study the protective effect and mechanism of iPSC⁃MSCs on cartilage matrix in knee oste⁃ oarthritis (KOA) patients in vitro. Methods : Cartilage tissues removed from KOA patients with joint replacement surgery were collected and subjected to tissue and cellular experiments , respectively. Cartilage tissue was cut into small pieces and randomly divided into a control group , an IL⁃1β ( 10 ng/ml) induction group , and iPSC⁃MSCs 96 h and then co⁃cultured with different amounts of iPSC⁃MSCs ( 1 × 104 , 1 × 105 , 1 × 106 ) cells for 72 h. For in tissues , the pathological changes of isolated cartilage tissues were examined by HE staining. The levels of ADAMTS⁃4 , ADAMTS⁃5 , and type II collagen expression were analyzed by immunohistochemistry , while the levels of MMP13 , IL⁃6 , and IL⁃10 in culture supernatants were detected by ELISA kits. The 2 to 5 generations of chondrocytes , which were extracted from cartilage tissue of KOA patients , were stimulated with IL⁃1β ( 10 ng/ml) for 48 h and then co⁃cultured with different concentrations of iPSC⁃MSCs ( 1 × 104 , 1 × 105 , 1 × 106 ) cells for 72 h. Immunofluorescence and Western blot detected the expression of RUNX2 , ADAMTS⁃4 , and ADAMTS⁃5 in chondrocytes. Results : Comparison with the control group , in the IL⁃1β⁃induced group , the levels of RUNX2 , ADAMTS⁃4 , and ADAMTS⁃5 increased , the level of type II collagen decreased , the levels of MMP⁃13 and IL⁃6 in the culture supernatant increased ( P < 0. 05) , and the level of IL⁃10 decreased ( P < 0. 05) ; Compared with the IL⁃1β⁃induced decreased the expression of RUNX2 , ADAMTS⁃4 , and ADAMTS⁃5 , promoted type II collagen expression and elevated IL⁃10 levels. Conclusion iPSC⁃MSCs inhibited ADAMTS⁃4 and ADAMTS⁃5 expression in vitro , reduced cartilage extracellular matrix degradation , and played a role in articular cartilage protection.

Objective: To establish and evaluate the pyroptosis model of rat chondrocytes induced by tumor necrosis factor-α(TNF-α). Methods: A two-step enzymatic digestion method was used to obtain rat articular chondrocytes, inverted phase contrast microscope was used to observe the morphological structure of chondrocytes. Toluidine blue staining and type Ⅱ collagen(Col Ⅱ) staining were used to identify chondrocytes. Different mass concentrations of TNF-α(5, 10, 20, 40 ng/ml) were used to establish the pyroptosis model with TNF-α(0 ng/ml) as the control group. Cell viability was detected by CCK-8 method and proteins of pyroptosis signal were detected by Western blot. The levels of IL-1β and IL-18 in cultured supernatants were examined by ELISA kits. The expression of gasdermin D(GSDMD) in chondrocytes was detected by immunofluorescence. Scanning electron microscope was used to observe the morphological changes of chondrocyte. Results: Compared with the control group, the cell viability of rat chondrocytes gradually decreased with the increase of TNF-α concentration and the expression of nucleotide-binding and oligomerization domain-like receptor containing protein 3(NLRP3), caspase-1, GSDMD and Phospho-NF-κB p65(P-p65) proteins increased. Furthermore, TNF-α(20 ng/ml) could up-regulate the expression of matrix metalloproteinase-13(MMP-13), the fluorescence expression of GSDMD and the levels of IL-1β and IL-18 while the expression of Col Ⅱ was distinctly reduced. What′s more, the articular chondrocytes were swollen,and the microstructure was destroyed. Conclusion TNF-α(20 ng/ml) can cause the swelling and death of rat chondrocytes, degradation of cartilage matrix and activation of pyroptosis signaling pathway. The pyroptosis model of rat chondrocytes was successfully established.

Objective: To establish a method for isolation and culture of primary endothelial cells from non-human primate coronary arteries, and to provide a cell model for the study of human coronary endothelial cells. Methods: The coronary arteries of macaca mulattas were separated aseptically. The primary endothelial cells were separatedviatissue adhesion after collagenase digestion. CD31 positive cells were detected and sorted by flow cytometry to determine the purity of endothelial cells. After stimulation with prostaglandin E2(PGE2), the cellular viability and proliferation ability of primary coronary endothelial cells from macaca mulattas were evaluated by high-content cell imaging and CCK-8 assay, and the migration ability and tube function of primary coronary endothelial cells from macaca mulattas were measured by Transwell method and Matrigel glue method, respectively. Results: The confluence percentage of primary coronary artery cells of macaca mulattas was about 80% after 10-14 daysin vitroculture, and the cellular morphology was irregular polygons and paver shape. The purity of endothelial cells was about 31.7% by flow cytometry. After sorting, the purity of endothelial cells was confirmed by flow cytometry, which was more than 95%. PGE2could significantly up-regulate the proliferation, migration and tube formation abilities of primary coronary endothelial cells of macaca mulattas. Conclusion This study successfully established the isolation and culture method of primary coronary endothelial cells from macaca mulattas, and proved that it could be used as anin vitrocell model to simulate human coronary endothelial cells through functional studies.

Objective: To compare the efficiency of different methods for extracting rhesus monkey lung fibroblasts and their effects on functions, so as to provide a method for obtaining primary lung fibroblasts that are closer to human fibroblasts. Methods: Two extraction methods for rhesus monkey lung fibroblasts were used, direct tissue block adhesion method and collagenase combined digestion with tissue block adhesion method. The cell morphology was observed with the inverted microscope, the purity of isolated rhesus monkey lung fibroblasts was identified by immunofluorescence, cell viability was detected by CCK-8, the expression of α-SMA was detected by flow cytometry and the effect of long-term in vitro culture on cell apoptosis was detected by apoptosis kit. Western blot was used to detect the expression of α-SMA protein. Results: The combined digestion with collagenase and tissue block adhesion method could see small and bright cells crawling out in 24 hours, and cells could be seen crawling out in a large area after 48 hours. The cells were in a long spindle shape, after 4 days to 5 days, a single layer of cells could be formed. Identified by immunofluorescence, all cells expressed α-SMA. Tissue adhesion method showed small and bright cells crawling out after 72 hours. After 4 days to 5 days, the cells crawled out in a small area and showed a long spindle shape. After a week, the cells crawled out in a large area and formed a single layer of cells and the cells are all expressed α-SMA by immunofluorescence. The experimental results showed that the cell viability of the cells crawled out by the collagenase digestion method was significantly higher than that of the tissue adhesion method. After TGF-β1 stimulates the cells, the cells extracted by collagenase digestion method proliferated faster and expressed α-SMA more obviously. Conclusion Both methods can isolate rhesus monkey lung fibroblasts in vitro, but the collagenase digestion method extracts cells in a shorter time and in better condition. The expression of related proteins is more stable after stimulation by stimulants, which is an effective method to obtain rhesus monkey lung fibroblasts, and it is also an effective method to obtain primary lung fibroblasts that are closer to human.

Abstract: To observe the histomorphological features of the hypothalamic-pituitary-adrenal axis in macaques to provide a reference for simulating the physiological functions and pathological responses of the human hypothalamic-pituitary-adrenal axis. Methods: After euthanasia of macaques, hypothalamus, pituitary and adrenal tissues were removed intact, fixed by PFA, and paraffin sections and frozen sections were prepared; the basic structure and cellular distribution were observed by HE staining; the secreted hormones and receptors were detected by immunohistochemistry; the effects of staining in frozen and paraffin sections were compared, and the cellular composition of some hypothalamus tissues was identified. Results: The hypothalamic region was hollow and funnel-shaped, the pituitary gland resembles a pea, and the right and left adrenal glands were located between the liver and kidneys; HE staining showed that the hypothalamic region was mainly composed of neurons and microglia, the pituitary gland was divided into neuro-pituitary and adeno-pituitary, and the adrenal gland was composed of cortex and medulla; immunohistochemical results showed that the hypothalamus secretes CRH and expresses GR, the pituitary gland secretes ACTH and expresses CRHR1 and GR, and the adrenal gland expresses ACTHR; immunofluorescence of frozen sections better showed that the hypothalamus contains neurons and microglia. Conclusion In this study, sections of hypothalamus, pituitary and adrenal gland tissues from macaques were successfully produced, and the relevant anatomical and morphological features were observed and examined, which provided a reference method for simulating the physiological and pathological responses of the human hypothalamic-pituitary-adrenal axis.

Objective: To investigate the therapeutic and immunomodulatory effects of human umbilical cord-derived mesenchymal stem cells (hUC-MSCs) on adjuvant arthritis(AA) rats. Methods: Twenty-four male Sprague-Dawley(SD) rats were established with AA by complete Freund's adjuvant method.They were randomly divided into model group and hUC-MSCs group (2×10⁶ cells/mL, 5×10⁶ cells/mL, tail vein injection), and the Yisaipu group (2.8mg/kg, subcutaneous injection), 6 rats in each group.Another 6 male SD rats were used as the control group.After the model was established, the body weight and paw volume were recorded weekly, the whole body score and the arthritis index score were calculated, and the joint swelling number was calculated.The animals were sacrificed after d35, the weight of thymus and spleen were weighed, and the corresponding index was calculated, the histopathological changes of the ankle joint were observed by HE staining.The percentages of CD4⁺ CD44⁺ T cell and CD4⁺ CD62L⁺ T cell were detected by flow cytometry.The levels of TNF-α, IL-1β in the serum of AA rats were detected by ELISA. Results: hUC-MSCs relieved paw volume, the whole body score and arthritis index score, and the joint swelling number in AA rats (F=20.573, 89.092, 14.161, 10.914, all P<0.01). hUC-MSCs reduced thymus index[(0.120±0.032), (0.120±0.031)] and spleen index[(0.250±0.070), (0.240±0.018)] (F=6.339, 4.105, all P<0.01), improved structural damage of ankle joint.hUC-MSCs could regulate the percentage of T cell subsets(CD4⁺ CD62L⁺ )[(7.0±1.4)%, (7.9±2.2)%], (CD4⁺ CD44⁺ )[(15.0±3.6)%, (12.0±1.9)%] in spleen (F=6.331, 12.719, all P<0.01), and down-regulate the levels of TNF-α [(172.0±13.0)ng/L, (150.0±12.0)ng/L] and IL-1β [(75.0±36.0)ng/L, (74.0±20.0)ng/L] in serum (F=8.221, 3.581, all P<0.05) of AA rats by tail vein injection. Conclusion hUC-MSCs can promote the treatment of AA rats by regulating the function of T cells.

Objective To establish and evaluate a rat model presenting symptoms of arthritis-hypertension disease (AHD).Methods A total of forty healthy 5-6 week-old male SD rats were used in this study.Hypertension was induced by constriction of the left renal artery by two kidney one clip (2K1C) with a 0.25 mm silver clamp, and AHD model was established by injecting 0.1 mL complete Freund adjuvant to the left hind paw.Tail artery pressure was measured with a non-invasive blood pressure measurement system.The degree of swelling in the non-inflammatory joint of rats was measured with a paw volume measuring instrument, the arthritis index and incidence of inflammation were evaluated.The rats were sacrificed on the 35th day.The thoracic aorta, ankle joint and spleen tissues were examined by pathology using HE staining.Results The joint of AHD model rat was significantly swollen, extensive synovial cell hyperplasia, inflammatory cell infiltration, vascular pannus formation, and bone and cartilage destruction.The number of germinal centers in spleen was increased, and a large number of lymphocyte infiltration, diffuse proliferation of white pulp, and red pulp infiltration were present.The arthritis index, incidence of inflammation and histopathological scores of the joint and spleen were significantly higher than adjuvant arthritis (AA) rats;meanwhile, the blood pressure of AHD model rat was significantly increased, the thickness and cross-sectional area of thoracic aorta were significantly increased, while the lumen diameter was significantly reduced.The blood pressure and vascular injury were significantly increased or aggravated compared with the HT rats.Conclusions A rat model of arthritis-hypertension disease is successfully established by using complete Freund adjuvant intradermal injection to the footpad and surgery to narrow the left renal artery.