微囊肝细胞大规模培养

发布时间：2018-04-01 12:27

本文选题：微囊肝细胞　切入点：转瓶转速　出处：《浙江大学》2008年博士论文

【摘要】： 背景和目的:观察微囊HepA、HepG_2细胞转瓶培养时转速、细胞密度对细胞增殖的影响。方法:一步法AC微囊包裹HepG_2及第25代HepA细胞,随机分为2大组:试验组为HepA细胞组;对照组为HepG_2细胞组。每组按包埋的细胞密度、转瓶转速分8小组,所包埋的细胞密度分别为:1.0X10~6、2.0X10~6,转瓶转速分别为0RPM、2RPM、3RPM、4RPM,初始细胞数目均为1.0X10~7,动态观察并测量各组聚集体大小、数目、细胞数目变化,Elivision plus免疫组化法检测各组Ki67表达情况。结果:各组每个微囊内聚集体平均个数从第2天的1.1-4.5个迅速增至第8天3.2-13.1个,每个微囊内聚集体平均最大径从第2天的23.8-68.7um迅速增至第8天44.9-128.9um,之后增长减缓,至第14天聚集体个数达5.4-18.5个,最大径达50.6-183.4um。无论任何时间点,静止培养组Ki-67强阳性百分率均最低(d_2 0-5%,d_7 40-45%,d_(12)10-20%),弱阳性百分率最高(d_2 70-75%,d_7 45-50%,d_(12)75-80%);4RPM组Ki-67强阳性百分率均最高(d_2 65-70%,d_7100%,d_(12)75-80%),弱阳性百分率最低(d_2 30-35%,d_7 0%,d_(12)20-25%); 各组第1天均处于相对静止期,第2天进入对数增长期,第8天达到高峰,静止培养组、2RPM组、3RPM组、4RPM组扩增倍数分别达到初始数目的4.02-4.79倍、17.69-19.79倍、22.95-25.83倍、35.11-36.87倍,之后增长速度渐趋缓慢,第8天至第10天,各组扩增倍数不及第8天的五分之一,第14天各组扩增倍数分别达到初始数目的近5.87-6.42倍、23.86-24.88倍、30.18-30.89倍、43.06-43.92倍。结论:微囊HepA、HepG_2细胞各组随转速增加,每个微囊内聚集体平均最大径、平均个数明显增多;Ki-67强阳性百分率逐渐升高,弱阳性百分率逐渐降低;细胞增长速度明显加快(P均等于0.00);但均与包裹细胞密度关系不大(P均大于0.05);各组在各个时间点每个微囊内聚集体平均最大径、平均个数、Ki-67强阳性百分率、弱阳性百分率、细胞数目无明显区别(P均大于0.05)。背景和目的:观察微囊HepA、HepG_2细胞转瓶培养时转速、细胞密度对细胞功能活性的影响。方法:分组方法同第一部分,Elisa动态检测各组白蛋白合成量,全自动生化仪分析尿素合成量,HPLC法检测安定转化功能,半定量RT-PCR检测各组CYP450-3A4、CYP450-2E1、Cx32表达量。结果:微囊HepA、HepG_2各组白蛋白合成量从第2天的0.14-0.71pg/cell/h迅速增加,至第10天达到高峰0.60-3.50pg/cell/h,而后迅速衰减,第14天各组均检测不到白蛋白合成。尿素合成功能微囊HepA、HepG_2组分别从第2天的0.09-0.23pg/cell/h、0.16-0.62pg/cell/h迅速增至第10天达高峰0.46-4.92pg/cell/h、0.60-6.68pg/cell/h,而后迅速衰减,至第14天,仅0.13-2.51pg/cell/h、0.19-3.72pg/cell/h。安定转化功能HepA、HepG_2组分别从第2天的0.41-2.42pg/cell/h、0.12-0.69pg/cell/h迅速增至第10天1.67-11.87pg/cell/h、0.71-5.08pg/cell/h之后迅速衰减,第12天仅0.62-5.77pg/cell/h、0.24-1.72pg/cell/h,第14天均检测不到。半定量RT-PCR:静止培养各组细胞CYP3A4、CYP2E1、Cx32 Ct比值任何时间点均小于1;旋转培养微囊HepA各组CYP3A4在任何时间点均大于1;Cx32、CYP2E1第7天比值均大于1,第12天Cx32仅4RPM组大于1,CYP2E1 3RPM、4RPM组均大于1;微囊HepG_2各组Cx32在任何时间点均小于1;CYP3A4、CYP2E1第2、7天比值均大于1,第12天各组CYP3A4比值均小于1,CYP2E1 3RPM组、4RPM组均大于1。结论:各组随转速增加,细胞尿素、白蛋白合成量、安定清除量,Cx32、CYP3A4、CYP2E1表达量均明显增多(P均等于0);但均与包裹细胞密度关系不大(P均大于0.05);微囊HepA各组在各个时间点的尿素合成量、安定清除量、Cx32、CYP3A4表达量均明显比HepG_2高(P均小于0.05),白蛋白合成量、CYP2E1表达量二者无明显区别(P=0.84)。
[Abstract]:Background and aim: To observe the effect of microcapsule HepA, HepG_2 cells in vase culture, and cell density on cell proliferation.
Methods: one-step AC microencapsulated HepG_2 and the 25 generation of HepA cells, were randomly divided into 2 groups: experimental group HepA cells group; control group HepG_2 group. Each group according to embedded cell density, spin the bottle speed divided into 8 groups, the entrapped cell density were 1.0X10~6,2.0X10~6, turning speed respectively 0RPM, 2RPM, 3RPM, 4RPM, initial cell number was 1.0X10~7, the number of dynamic observation and measured the aggregate size, and cell number changes, Elivision plus immunohistochemical method to detect the Ki67 expression.
Results: the average number of each microcapsule aggregates from 1.1-4.5 second days to eighth days 3.2-13.1 quickly, the average maximum diameter of each microcapsule aggregates from second days to eighth days 44.9-128.9um 23.8-68.7um quickly, after the growth slowed down, the number of aggregates to fourteenth days reached 5.4-18.5, the maximum diameter of up to 50.6-183.4um.
At any point in time, the static culture group Ki-67 positive percentage was the lowest (d_2 0-5%, d_7 40-45%, d_ (12) 10-20%), weakly positive for the highest percentage (d_2 70-75%, d_7 45-50%, d_ (12) 75-80%); group 4RPM had the highest percentage of strong positive Ki-67 (d_2 65-70%, d_7100%, d_ (12) 75-80%), the lowest percentage of weak positive (d_2 30-35%, d_7 0%, d_ (12) 20-25%);
Every first days in a relatively quiescent period, second days into the logarithmic growth period, eighth days to reach the peak, the static culture group, 2RPM group, 3RPM group, 4RPM group was 4.02-4.79 times, times respectively, the initial number of 17.69-19.79 times, 22.95-25.83 times, 35.11-36.87 times, then the growth rate is slow, eighth to tenth days each. Less than eighth days of amplification multiples of 1/5, fourteenth days respectively were amplification times the initial number of nearly 5.87-6.42 times, 23.86-24.88 times, 30.18-30.89 times, 43.06-43.92 times.
Conclusion: microencapsulated HepA, HepG_2 cells were detected with the speed increasing, the average maximum diameter of each microcapsule collective cohesion, the average number increased significantly; Ki-67 positive percentage increased gradually, weak positive percentage gradually decreased; cell growth significantly faster (P = 0); but with little relationship (P package cell density were greater than 0.05); each group at each time point of each cohesive collective mean maximum diameter of microcapsules, the average number of Ki-67 positive percentage, weak positive percentage, no significant difference between the number of cells (P greater than 0.05).
Background and aim: To observe the effect of microcapsule HepA, HepG_2 cells in vase culture, and cell density on cell function activity.
Methods: grouping method and the first part, Elisa were used to dynamically detect the albumin synthesis in each group. The synthetic amount of urea was analyzed by automatic biochemistry analyzer. The transformation function of diazepam was detected by HPLC, and the expression of CYP450-3A4, CYP450-2E1 and Cx32 in each group was detected by semi quantitative RT-PCR.
Results: the albumin synthesis of microcapsules HepA and HepG_2 increased rapidly from second days to 0.14-0.71pg/cell/h, reaching the peak of 0.60-3.50pg/cell/h on the tenth day and then rapidly declined. On the fourteenth day, albumin synthesis was not detected in all groups.
Urea synthesis functional microcapsules HepA, HepG_2 group increased from second days 0.09-0.23pg/cell/h, 0.16-0.62pg/cell/h to tenth Tianda peak 0.46-4.92pg/cell/h, 0.60-6.68pg/cell/h, and then rapidly declined to fourteenth days, only 0.13-2.51pg/cell/h, 0.19-3.72pg/cell/h..
The anding transformation function of HepA and HepG_2 group increased rapidly from second days 0.41-2.42pg/cell/h to 0.12-0.69pg/cell/h for tenth days, and 1.67-11.87pg/cell/h rapidly declined after 0.71-5.08pg/cell/h. Twelfth days were only detected on 0.62-5.77pg/cell/h, 0.24-1.72pg/cell/h and fourteenth days.
Semi quantitative RT-PCR: CYP3A4 static culture, the cells were CYP2E1, Cx32 and Ct ratio at any point in time is less than 1; HepA group CYP3A4 rotation culture microcapsules at any point in time are greater than 1; Cx32, seventh days CYP2E1 ratios were more than 1, Twelfth days Cx32 group 4RPM is greater than 1, CYP2E1 3RPM, 4RPM group were higher than 1; microcapsule HepG_2 Cx32 in each group at any time point is less than 1; CYP3A4, CYP2E1 day 2,7 ratio was more than 1, Twelfth days each CYP3A4 ratio was less than 1 CYP2E1, 3RPM group, 4RPM group were more than 1.
Conclusion: each with increasing speed, the cell amount of albumin synthesis, urea, Cx32, diazepam clearance volume, CYP3A4, CYP2E1 expression was significantly increased (P = 0); but the cell density has little relation with parcel (P greater than 0.05); MC HepA groups at each time point of the urea contents, stability the amount of clearance, Cx32, CYP3A4 expression was significantly higher than HepG_2 (P < 0.05), the amount of albumin synthesis, the expression of CYP2E1 showed no significant difference between the amount of two (P=0.84).

【学位授予单位】：浙江大学
【学位级别】：博士
【学位授予年份】：2008
【分类号】：R329.2

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