Research On The Role And Mechanism Of Leptin In The Fibrosis And Hypertrophy Of Lumbar Ligamentum Flavum

Chapter 1 Isolation,cultivation,amplification and phenotype identification of ligamentum flavum cells in vitroObjective To establish the methods of the isolation,cultivation and amplification of ligamentum flavum?LF?cells in vitro.Methods Twelve patients with lumbar spinal canal stenosis?LSCS?underwent surgical treatment from April 2017 to July 2017 were enrolled in this study.The mean age of the patients was 62.8±3.6 years.For the control group,12 patients with lumbar disc herniation?LDH?was included.The mean age of the patients was 57.9±8.5 years.LF samples were obtained during the surgery from subjects underwent posterior decompression surgery,which were under the patient's informed consent without affecting the effect of the operation.The LF cells were isolated by collagenase-predigested explant cultures.Under an inverted phase microscope,cells were examined for migration,morphology and growth status.The MTT method was used to analyze the cell proliferation by measuring the optical density?OD?values.The second generation?P₂?cells were scraped to count.Expressions of vimentin and collagen I detected in P₂ cells by immunofluorescenee staining were used to confirm the phenotype of LF cells.Results Cell outgrowths were observed from LF tissue explants after about 10 days.Morphologically,LF cells varied widely in appearance,and arranged mainly as spindle shape and polygonal shape.After 20 days,the cells then turned to be fusiform in shape and confluent cultures.0.5×10⁶ cells could be obtained in each P₂generation culture flask.It took about 40 days from the P₀ to the P₂ generation of LF cells.There was no statistical difference between the two study groups about increment time of LF cells.The positive results of expression of vimentin and collagen I detected by immunofluorescenee stain confirmed that the P₂ cells were typical LF cells.Conclusion 1.Collagenase predigestion explant method offers copious primary LF cells with good cellular characters,it is an ideal culture system for human LF cells.2.LF cells from the two study groups have the same capacity of growth and proliferation in vitro.3.LF cells in vitro cultured present fibroblast-like phenotypes.Chapter 2 The role of Leptin in the fibrosis and hypertrophy of lumbar ligamentum flavumObjective To detect the exprssion of Leptin m RNA and protein in lumbar ligamentum flavum?LF?,and explore its role in the LF hypertrophy and fibrosis.On this basis,the recombinant human Leptin?rh Leptin?in the culturing LF cell was added and then the collagen expression were detected to clarify the role of leptin in the process of LF fibrosis.Methods This study included two parts: the tissue and cell experiments.LF specimens were divided into two groups according to different diseases: LSCS group?patients with lumbar spinal canal stenosis,n=12?and LDH group?patients with lumbar disc herniation,n=12?.The LF specimens were obtained during the surgery.The morphologic changes and fibrosis score of LF were assessed by H&E and Masson's trichrome staining,respectively.The location and expression of Leptin in LF tissues were determined by Immunohistochemical Staining,RT-PCR and Western Blotting.Then,the LF cells were cultured and exposed to rh Leptin.Collagen I and III expression were detected and used as fibrosis markers.Results Patients in two groups showed similar outcomes regarding age,gender,level of LF tissue?P>0.05?.The LF thickness and fibrosis score in LSCS group were significantly higher than those of LDH group?P<0.05?.Histologically,it was found that the LF was disorganized and presented fibrotic changes.The LF thickness was positively correlated with the fibrosis score.Leptin was detected in the hypertrophied LF and its expression was substantially increased in LSCS group and positively correlated with LF thickness and fibrosis score?P<0.05?.Moreover,our in vitro experiments revealed that rhleptin treated LF cells elevated the expression of collagen I and III.Conclusion 1.The hypertrophic LF presents a typical fibrosis change.2.Leptin expression increases significantly in LF samples from LSCS and exhibits a positive correlation with the LF thickness and fibrosis score.3.Leptin promotes the collagen expression in LF cells.4.The increased expression of Leptin in human LF from patients with LSCS may play an important role in the pathogenesis of the LF fibrosis and hypertrophy.Chapter 3 Research on the role and mechanism of Leptin regulating NF-?B signaling pathway in the fibrosis and hypertrophy of lumbar ligamentum flavumObjective To clarify the role and mechanism of Leptin regulating NF-?B signaling pathway in the fibrosis and hypertrophy of lumbar ligamentum flavum.Methods On the basis of Experiment one and Experiment two,P2 generation of LF cells were collected.Different Lepin concentrations of cell culture medium were prepared,and the LF cells were grouped and cultured for 48 hours.The expression of p65 and IL-6 m RNA and protein in the LF cells of different groups was detected by RT-PCR,Western-blotting and ELISA.Finally,The inhibitor of NF-?B signaling pathway?Bay 11?was added into the culture medium with high concentration of Lepin,and then the expression levels of IL-6 m RNA and protein were examined.Results After treatment with leptin,there was a rapid increase in p65 nucleoprotein levels.In addition,leptin administration elevated IL-6 m RNA and protein expression?P < 0.05?.P65 was a crucial adaptor for leptin induced IL-6,as the upregulated IL-6 expression induced by leptin was significantly attenuated by an NF-?B inhibitor Bay 11.Conclusion Lepin can stimulate the expression of IL-6 by activating the NF-?B signaling pathway and induce local inflammatory response,which may be an important mechanism leading to the LF fibrosis and hypertrophy.