摘要
目的:建立肠系膜微淋巴管内皮细胞的培养技术,观察不同体积分数的休克淋巴液对大鼠肠系膜微淋巴管内皮细胞形态的影响。方法:实验于2004-02/07在河北北方学院病理生理学教研室及实验中心细胞培养室完成。选择健康雄性Wistar大鼠,体质量分别为220～300g和50～80g。实验方法:①无菌条件下制备大鼠重症失血性休克模型,引流肠系膜淋巴液或收集门静脉血;同时另取大鼠,引流正常淋巴液或正常门静脉血。②从动物种类、培养基、植块方法、胎牛血清浓度等方面对植块培养法进行改良,进行肠系膜微淋巴管内皮细胞原代培养并传代。实验分组:分为6组:胎牛血清组:培养液为DMEM+体积分数为0.1的胎牛血清;正常淋巴液组:培养液为DMEM+正常淋巴液;休克淋巴液组:培养液为DMEM+体积分数为0.04,0.06,0.08,0.10的休克淋巴液;正常血浆组:培养液为DMEM+正常血浆;休克血浆组:培养液为DMEM+休克血浆;无血清对照组:培养液为DMEM。实验评估:①光镜下观察大鼠肠系膜微淋巴管内皮细胞原代培养及传代情况。②光镜、扫描电镜及透射电镜下观察不同体积分数的休克淋巴液及不同作用时间(4,8,12h)对肠系膜微淋巴管内皮细胞形态及超微结构的影响。结果:①光镜下肠系膜微淋巴管内皮细胞原代培养及传代情况:内皮细胞呈扁平的梭形或多边形,大小均匀,胞核清晰,呈卵圆形。细胞生长融合形成单层后,呈典型的鹅卵石或铺路石样镶嵌状排列生长。②光镜下在休克淋巴液中肠系膜微淋巴管内皮细胞形态:体积分数为0.04的休克淋巴液作用4h时细胞逐渐收缩、变圆直至脱落漂浮,随着休克淋巴液体积分数增加及作用时间延长,细胞损伤逐渐加重,体积分数为0.08的休克淋巴液作用4h时核旁出现空泡,体积分数为0.10的休克淋巴液作用12h时细胞裂解成碎片。其他各组作用12h肠系膜微淋巴管内皮细胞形态无显著改变。③扫描电镜下在休克淋巴液中肠系膜微淋巴管内皮细胞形态:体积分数为0.04的休克淋巴液作用4h部分细胞逐渐收缩离壁,细胞间隙增大、细胞边缘的突起拉长、缩短、断裂,作用8,12h可见凋亡小体。其他各组作用12h肠系膜微淋巴管内皮细胞形态无显著改变。④透射电镜下在休克淋巴液中肠系膜微淋巴管内皮细胞形态:体积分数为0.04的休克淋巴液作用4h细胞膜形态不规整,胞浆内质网等膜性细胞器扩张,核形态不规则,作用8h线粒体呈球形扩张,细胞核逐渐出现固缩、似细胞凋亡改变,核周腔变宽;体积分数为0.08的休克淋巴液作用时间8h细胞内出现囊泡,内质网不同程度扩张,线粒体主要表现为浓缩、深染等改变,细胞膜边界不清,出现起泡、破碎等现象。其他各组作用12h肠系膜微淋巴管内皮细胞形态无显著改变。结论:成功建立了肠系膜微淋巴管内皮细胞的培养技术;休克淋巴液可导致肠系膜微淋巴管内皮细胞形态及超微结构损伤。
AIM： To develop the method for isolation and primary culture of mesentery micro-lymphatic endothelial cells （MMLECs） of rats, and observe the effects of shock lymph fluid at different volume fractions on the morphology of MMLECs. METHODS： The experiment was conducted in the Department of Pathophysiology and Cell Culture Laboratory of Hebei North University from February to July 2004. Healthy male Wistar rats of 220-300 g and 50-80 g were selected. （1）The model of severe hemorrhagic shock was established in sterile condition and mesentery lymph fluid or portal vein blood was collected. Meanwhile, normal mesentery lymph fluid, or normal portal vein blood of other rats was harvested. （2）The tissue explants attachment culture method was improved in experiment animal, culture medium, tissue explants method, and fetal bovine serum （FBS） concentration. The MMLECs were primarily cultured and divided into 6 groups： FBS group （DMEM ＋ 0.1 volume fraction FBS）; normal lymph fluid group （DMEM ＋ normal lymph fluid）; shock lymph fluid group （DMEM ＋ 0.04, 0.06, 0.08, and 0.10 volume fractions shock lymph fluid）; normal plasma group （DMEM ＋ normal plasma）; shock plasma group （DMEM ＋ shock plasma）; serum-free control group. The primary culture and passage of MMLECs were observed under light microscope; the effects of shock lymph fluid at different volume fractions and action time （4, 8, and 12 hours） on the morphology and ultrastructure of MMLECs were observed under light, transmission electron microscope and scanning electron microscope. RESULTS： （1）In the light microscope study, the MMLECs cultured primarily presented in flat fusiform shape or polygon, with even size, clear orbicular-ovate nucleus. When the cells grew and formed monolayer, they were typical cobbly arranged. （2）After 4 hours culture in 0.04 volume faction shock lymph fluid, MMLECs gradually contracted, changed to round until shed and floated observed under light microscope. With the increase of volume fraction and action time of lymph fluid, cell damage was aggravated. After cells were cultured in 0.08 volume fraction shock lymph fluid for 4 hours, vacuoles were found around nucleus, and in 0.10 volume fraction shock lymph fluid for 12 hours, cells disrupted into pieces. However, no apparent changes were found in MMLECs in other groups within 12 hours. （3）Under scanning electron microscope, some cells were found gradually contracted and away from the wall after cultured in 0.04 volume fraction shock lymph fluid for 4 hours; cell spaces were enlarged with extended, shortened, and broke process; after cultured for 8, and 12 hours, apoptotic bodies were observed. However, there was no obvious change in MMLECs in other groups within 12 hours. （4）In the transmission electron microscope study, after MMLECs were cultured in 0.04 volume fraction shock lymph fluid for 4 hours, the cell membrane was irregular, endocytoplasmic reticulum （ER） and other membrane cell organs were extended, with irregular nucleus; after for 8 hours, mitochondria was expanded globularly, cell nucleus was pyknosis, and the pednuclear space was widened; after cells were cultured in 0.08 volume fraction shock lymph fluid for 8 hours, vesica was found in cells, and ER continued to expand; mitochondria was condensed, deeply stained with unclear membrane edge, and pieces. No apparent changes were found in MMLECs in other groups within 12 hours. CONCLUSION： The pdmary culture technique of MMLECs is established successfully. Shock lymph fluid could damage the morphology and ultrastructure of MMLECs.
出处
《中国组织工程研究与临床康复》
CAS
CSCD
北大核心
2007年第27期5424-5428,共5页
Journal of Clinical Rehabilitative Tissue Engineering Research
基金
国家自然科学基金资助项目(30370561)
河北省自然科学基金资助项目(C2004000649)~~
