Effect Of Recombinant Human Endostatin On Cell Proliferation Of Human Keloid Fibroblasts And Its Mechanisms

Objective To observe the effects of recombinant human endostatin（rhEndostatin） oncell cycle of keloid-derived fibroblasts（KFs）. And investigate the effects of rhEndostatinon the expression of the cell proliferation in KF_s. Investigate the role of cellular andmolecular mechanisms on cell, and provid experimental data for the development ofnew therapeutic interventions and targets of keloid.Methods①HE staining of the keloid: keloid after surgery to take4%paraformaldehyde, gradient alcohol dehydration, xylene, and embedded in paraffin,sliced; hematoxylin-eosin （HE） staining; neutral gum Fengpian; microscope observationand radiography.②Culture of KFs in vitro: Seven cases of keloid tissue （abdomen, twocases of ear lobe of four cases, one cases of thigh） from Anhui Medical UniversityAffiliated Hospital of Plastic Surgery keloid patients. Into fiber cells in primary cultureaccording to the literature method, to be passaged in cell fusion, experimental cells wereused in the third generation of the logarithmic phase of KFs.③Cells were identified: thecultured cells were identified by cell morphology and cell immunohistochemicalstaining techniques to its cell properties.④Cell proliferation detection:Take the thirdgeneration of KFs, with different doses rhEndostatin (6.25×10^-6,1.25×10^-5,2.50×10^-5,5.00×10^-5,1.00×10^-4mg/L) for24h,48h,72h. Tetrazolium salt colorimetric（MTT） to detect dose rhEndostatin the inhibitory rate of KFs the calculationrhEndostatin role of KFs50%inhibitory concentration (50%Inhibition concentration,IC₅₀),In this study, the IC₅₀value was5.00×10^-5mg/L.⑤Cell cycle detection: KF_scultured in passage2seeded in6-well plates. Cell is divided into control group, rhEndostatin group (5.00×10^-5mg/L),5-fluorouracil （5-FU） group （0.5mg/mL）.Serum-free medium for24h, cell cycle synchronization, followed by replacingthe culture medium containing20%serum, and the corresponding join rhEndostatin(final concentration of5.00×10^-5mg/L), and5-FU （final concentration of0.5mg/mL）,and continue for48h, cells were collected, detected cell cycle by FCM.⑥Cellcycle-related gene expression: Immunohistochemical staining to detect expression ofKFs of5.00×10^-5mg/L rhEndostatin48h on p⁵³, proliferating cell nuclear antigen（preliferation cell nuclear antigen, PCNA）, cyclinD1and cyclin-dependentkinase-4（cyclin-dependent kinase4, CDK₄）.Results①HE staining: Microscope keloid dermal inflammatory cell infiltration, alarge number of fibroblast proliferation and collagen deposition; Part of the collagenfibers arranged in disorder, were bundle or nodular formation of fibrous cord, theoccurrence of hyalinosis, Suggesting that surgical specimens for the keloid.②Theculture of KFs in vitro:Organize block inoculation after7⁸d, the surrounding areahas seen a few spindle cells, followed by the cells gradually increased and the tissueblock radial growth; Passage2cultured cell monolayers arranged in the form of uniform,elongated spindle refraction.③Identification results of KFs: Cell vimentinimmunohistochemical staining shows cytoplasm showed brown.④Effect ofrhEndostatin on the cell proliferation of KFs: In addition to the6.25×10^-6mg/Lconcentrations, Dose groups rhEndostatin can significantly inhibit the proliferation ofKFs, each group KFs inhibit the rate of two compared with a statistically significantdifference （P <0.05） inhibitory effect was certain with rhEndostatin dose and timedependencies. Of which,5.00×10^-5mg/L and1.00×10^-4mg/L group rhEndostatin for48h, cell growth is slow, smaller size, reduce the number of disordered.⑤Effect ofrhEndostatin on the cell cycle of KFs: Results of detection and analysis by FCMshowed that,control group the KFs cell cycle is the main stay in the S phase （42.21± 3.32）, G₂/M phase cells were （19.70±3.64）; After to join rhEndostatin (5.00×10^-5mg/L), Cells in G1phase by the control group （36.30±4.40） to （65.09±3.20）, whilethe S and G₂/M phase cells were significantly reduced, respectively （12.86±2.14） and（12.78±3.40）.⑥Effect of rhEndostatin on p⁵³, PCNA, CyclinD₁and CDK4expressionof KFs: p⁵³, PCNA, CyclinD1and CDK4-positive staining showed brown or finegranular, in addition to CDK4in the nucleus and cytoplasm, the rest are located in thenucleus. The results show, p⁵³, PCNA, CyclinD1and CDK4were strongly positiveexpression in KFs were79.32%,89.45%,61.13%and82.34%, after treatment for48h,5.00×10^-5mg/L rhEndostatin, p⁵³, PCNA, CyclinD₁and CDK₄protein expressiondecreased, respectively,16.18%,12.23%,24.98%and15.26%.Conclusions rhEndostatin has the role of inhibition of KFs proliferation, itsmechanism may be rhEndostatin of inhibition of p⁵³, CDK₄, CyclinD1and PCNAexpression.