多房棘球蚴组织蛋白体外刺激肝星状细胞变化的实验观察

Experimental observation on the changes of hepatic stellate cells stimulated in vitro with tissue protein of Echinococcus multilocularis

目的观察多房棘球蚴组织蛋白体外刺激肝星状细胞(HSC)‑LX2后的细胞形态、胞内脂滴和细胞分泌α平滑肌肌动蛋白(α‑SMA)的变化。方法无菌取多房棘球蚴保种小鼠体内的多房棘球蚴组织,超声裂解法提取多房棘球蚴组织蛋白,以0.125 mg/ml终浓度加至体外培养的HSC‑LX2中刺激24、48、72、96和120 h后,取细胞进行油红O染色,光学显微镜和荧光显微镜下观察细胞形态和胞内脂滴分布。提取刺激48、72、96和120 h后的HSC‑LX2总蛋白,Western blotting检测细胞内α‑SMA的相对表达量变化。结果光学显微镜下可见,多房棘球蚴组织蛋白刺激24~96 h后,HSC‑LX2细胞形态由多边形逐渐变为长梭形,胞内脂滴数量增加且脂滴分布在细胞核周围,脂滴颗粒逐渐变大,颜色由浅黄色变为红色;刺激120 h后,绝大部分细胞都变成长梭形,多数细胞内脂滴数量有所减少且体积变小,脂滴由红色变为黄色。荧光显微下可见,刺激不同时间的HSC‑LX2胞内有红色荧光的脂滴颗粒围绕在细胞核周围,荧光信号不随着照射次数的增加而降低。Western blotting分析结果显示,HSC‑LX2胞内α‑SMA相对表达量随刺激时间延长逐渐升高,刺激至96 h时最高,为4.846±0.741,至120 h时下降至2.420±0.349,但均高于对照组的(t=8.988、7.045,均P<0.01)。结论多房棘球蚴组织蛋白体外可促进HSC‑LX2活化,细胞形态发生改变,胞内脂滴数量和体积均先升高后下降,胞内α‑SMA蛋白表达呈现先升高后降低的趋势。

Objective To observe the changes in morphology and intracellular lipid droplets andɑ‑smooth muscle actin(α‑SMA)expressed in the hepatic stellate cell(HSC)‑LX2 stimulated with Echinococcus multilocularis tissue protein in vitro.Methods The E.multilocularis tissue was collected from infected mice under aseptic conditions,with which the tissue protein was extracted by ultrasonic‑splitting.The protein extractant was added into HSC‑LX2 culture in vitro at a final concentration of 0.125 mg/ml to stimulate for 24,48,72,96 and 120 h,then the cells were stained with oil red O to observe the distribution of intracellular lipid droplets under light and fluorescence microscopes.The total protein of HSC‑LX2 cells was extracted after stimulation for 48,72,96 and 120 h to detect the changes of relative expression ofα‑SMA using Western blotting.Results Under the light microscope,the morphology of HSC‑LX2 changed gradually from polygonal to long spindle shape,and with prolonged stimulation with the E.multi⁃locularis tissue protein,the number of intracellular lipid droplets increased,and lipid droplets distributed around the nuclei.The color of lipid droplets gradually changed from light yellow to bright red.After 120 h,almost all HSC‑LX2 became long spindle‑shaped.The number and volume of lipid droplets decreased,and the color changed from red to yellow.Under fluorescence microscopy,red fluorescent lipid droplets could be seen surrounding the nucleus,and the fluorescence signal did not decrease with the increase of irradiation times.Western blotting showed that the relative expressionα‑SMA in HSC‑LX2 gradually increased with the prolongation of stimulation time,which peaked at 4.846±0.741 after 96 hours and decreased to 2.420±0.349 after 120 hours,but both were higher than that of the control group(t=8.988,7.045,both P<0.01).Conclusion The E.multilocularis tissue protein can promote the activation of HSC‑LX2 in vitro,inducing changes in cell morphology,the number and size of intracellular lipid droplets being raised first and then reduced,and the expression of intracellularα‑SMA protein showing a similar changing trend as well.