The Research On Activation Of Hepatic Stellate Cell By Cell Contact Between Macrophage And Hepatic Stellate Cell

Objective: To observe how cell contact can influence the activation of hepaticstellate cell through the co-culture of the direct contact and non-contact between therat hepatic stellate cells and macrophages.Methods:1. Grouping: Firstly, collagenase and protease sequential perfusionwere used to disperse cells of the liver of S-D rats. Then the rat hepatic stellate cellswere isolated by density gradient centrifugation. Nextly, using the transwell culturesystem to do the non-contact co-culture. Grouping: Group A: Inoculating1ml of HSCwhich is0.5×10~5per ml in the small chamber of the transwell culture system, whileinoculating2ml of macrophages which is3.0×10~5per ml in the big chamber. GroupB: Mixing the same amount of the HSC and macrophages as group A does andvaccinating1ml in the small chamber and2ml in the big chamber. Group C:inoculating2ml of macrophages which is3.0×10~5per ml in the big chamber of thetranswell culture system. Group D: Inoculating2ml of HSC which is0.5×10~5per mlin the big chamber of the transwell culture system, while inoculating1ml ofmacrophages which is3.0×10~5per ml in the small chamber. Group E: Mixing thesame amount of the HSC and macrophages as group C does, and vaccinating1ml inthe small chamber and2ml in the big chamber. Group F: Inoculating2ml of HSCwhich is0.5×10~5per ml in the big chamber of transwell culture system.2. Outcome measures:1ChecktheHSC’sa-SMAbyimmunocytochemicalmethod2Measure the secretion amount of Collagen type I, TGF-β1and PDGF in thesupernatant fluid from each group with ELISA3MeasurethecellProliferationfromeachgroupwithMTT3.Statistical approach: As for the average figures’ comparison of several samples,one-way ANOVA was adopted, while, LSD inspection was used for multiple comparison.P<0.05means there are statistical differences.Results:1. the positive expression of HSC’s a-SMA were found in group A、B、D、E and F.2. Collagen type I:Group A:36.364±1.892ng/ml;Group B:23.502±1.474ng/ml;Group C:10.293±1.724ng/ml;Group D:42.331±2.701ng/m l; GroupE:31.337±0.792ng/ml；Group F:35.544±0.907ng/ml;The amount from group B wasless than group A and the amount from group E was less than group D withstatistical differences (P <0.05), which indicated that the amount of Collagen type Ifrom the direct contact co-culture group was remarkably less than the non-contactco-culture group.TGF-β1:GroupA:301.115±15.448pg/ml;GroupB:406.182±19.224pg/ml;GroupC:265.140±10.521pg/ml;GroupD:383.555±9.246pg/ml;GroupE:435.356±5.842pg/ml;GroupF:279.726±8.517pg/ml；PDGF：GroupA：67.916±4.830pg/ml;GroupB:77.838±4.014pg/ml；GroupC:60.034±5.677pg/ml;GroupD:87.678±3.643pg/ml;GroupE:95.189±1.672pg/ml;GroupF:65.646±2.032pg/ml;The number of the two types of cytokine in group B was larger than group A and the number of the two types of cytokine in group E was larger than group D with statistical differences (P <0.05),which showed that the amount of PDGF and TGF-β1from the direct contact group were larger than the non-contact group.3. After48hours’ cultivation in the transwell culture system, the OD figure ofthe cells is:Group A:0.63±0.02;Group B:0.34±0.02;Group C：0.38±0.01;GroupD:0.72±0.08;Group E:0.30±0.01;Group F:0.48±0.05；The figure from group B is lessthan group A and the figure from group E is less than group D.There are statisticaldifferences among these groups.(P <0.05).After48hours’ cultivation, under thecondition of the same amount of cells at the beginning,the Proliferation of cells fromthe direct contact co-culture group is less than the non-contact co-culture group andthere are statistical differences among these groups.(P <0.05)Conclusion:1. The direct contact between HSC and macrophages could makethe secretion amount of PDGF and TGF-β1increase significantly.2. The direct contact between HSC and macrophages could make the secretionamount of Collagen type I reduce obviously.3. This experiment cannot verify that the direct contact between HSC andmacrophages could completely promote the activation of HSC.