本文選題：微囊肝細胞　切入點：轉(zhuǎn)瓶轉(zhuǎn)速　出處：《浙江大學》2008年博士論文

【摘要】： 背景和目的:觀察微囊HepA、HepG_2細胞轉(zhuǎn)瓶培養(yǎng)時轉(zhuǎn)速、細胞密度對細胞增殖的影響。 方法:一步法AC微囊包裹HepG_2及第25代HepA細胞,隨機分為2大組:試驗組為HepA細胞組;對照組為HepG_2細胞組。每組按包埋的細胞密度、轉(zhuǎn)瓶轉(zhuǎn)速分8小組,所包埋的細胞密度分別為:1.0X10~6、2.0X10~6,轉(zhuǎn)瓶轉(zhuǎn)速分別為0RPM、2RPM、3RPM、4RPM,初始細胞數(shù)目均為1.0X10~7,動態(tài)觀察并測量各組聚集體大小、數(shù)目、細胞數(shù)目變化,Elivision plus免疫組化法檢測各組Ki67表達情況。 結(jié)果:各組每個微囊內(nèi)聚集體平均個數(shù)從第2天的1.1-4.5個迅速增至第8天3.2-13.1個,每個微囊內(nèi)聚集體平均最大徑從第2天的23.8-68.7um迅速增至第8天44.9-128.9um,之后增長減緩,至第14天聚集體個數(shù)達5.4-18.5個,最大徑達50.6-183.4um。 無論任何時間點,靜止培養(yǎng)組Ki-67強陽性百分率均最低(d_2 0-5%,d_7 40-45%,d_(12)10-20%),弱陽性百分率最高(d_2 70-75%,d_7 45-50%,d_(12)75-80%);4RPM組Ki-67強陽性百分率均最高(d_2 65-70%,d_7100%,d_(12)75-80%),弱陽性百分率最低(d_2 30-35%,d_7 0%,d_(12)20-25%); 各組第1天均處于相對靜止期,第2天進入對數(shù)增長期,第8天達到高峰,靜止培養(yǎng)組、2RPM組、3RPM組、4RPM組擴增倍數(shù)分別達到初始數(shù)目的4.02-4.79倍、17.69-19.79倍、22.95-25.83倍、35.11-36.87倍,之后增長速度漸趨緩慢,第8天至第10天,各組擴增倍數(shù)不及第8天的五分之一,第14天各組擴增倍數(shù)分別達到初始數(shù)目的近5.87-6.42倍、23.86-24.88倍、30.18-30.89倍、43.06-43.92倍。 結(jié)論:微囊HepA、HepG_2細胞各組隨轉(zhuǎn)速增加,每個微囊內(nèi)聚集體平均最大徑、平均個數(shù)明顯增多;Ki-67強陽性百分率逐漸升高,弱陽性百分率逐漸降低;細胞增長速度明顯加快(P均等于0.00);但均與包裹細胞密度關(guān)系不大(P均大于0.05);各組在各個時間點每個微囊內(nèi)聚集體平均最大徑、平均個數(shù)、Ki-67強陽性百分率、弱陽性百分率、細胞數(shù)目無明顯區(qū)別(P均大于0.05)。 背景和目的:觀察微囊HepA、HepG_2細胞轉(zhuǎn)瓶培養(yǎng)時轉(zhuǎn)速、細胞密度對細胞功能活性的影響。 方法:分組方法同第一部分,Elisa動態(tài)檢測各組白蛋白合成量,全自動生化儀分析尿素合成量,HPLC法檢測安定轉(zhuǎn)化功能,半定量RT-PCR檢測各組CYP450-3A4、CYP450-2E1、Cx32表達量。 結(jié)果:微囊HepA、HepG_2各組白蛋白合成量從第2天的0.14-0.71pg/cell/h迅速增加,至第10天達到高峰0.60-3.50pg/cell/h,而后迅速衰減,第14天各組均檢測不到白蛋白合成。 尿素合成功能微囊HepA、HepG_2組分別從第2天的0.09-0.23pg/cell/h、0.16-0.62pg/cell/h迅速增至第10天達高峰0.46-4.92pg/cell/h、0.60-6.68pg/cell/h,而后迅速衰減,至第14天,僅0.13-2.51pg/cell/h、0.19-3.72pg/cell/h。 安定轉(zhuǎn)化功能HepA、HepG_2組分別從第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之后迅速衰減,第12天僅0.62-5.77pg/cell/h、0.24-1.72pg/cell/h,第14天均檢測不到。 半定量RT-PCR:靜止培養(yǎng)各組細胞CYP3A4、CYP2E1、Cx32 Ct比值任何時間點均小于1;旋轉(zhuǎn)培養(yǎng)微囊HepA各組CYP3A4在任何時間點均大于1;Cx32、CYP2E1第7天比值均大于1,第12天Cx32僅4RPM組大于1,CYP2E1 3RPM、4RPM組均大于1;微囊HepG_2各組Cx32在任何時間點均小于1;CYP3A4、CYP2E1第2、7天比值均大于1,第12天各組CYP3A4比值均小于1,CYP2E1 3RPM組、4RPM組均大于1。 結(jié)論:各組隨轉(zhuǎn)速增加,細胞尿素、白蛋白合成量、安定清除量,Cx32、CYP3A4、CYP2E1表達量均明顯增多(P均等于0);但均與包裹細胞密度關(guān)系不大(P均大于0.05);微囊HepA各組在各個時間點的尿素合成量、安定清除量、Cx32、CYP3A4表達量均明顯比HepG_2高(P均小于0.05),白蛋白合成量、CYP2E1表達量二者無明顯區(qū)別(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).

【學位授予單位】：浙江大學
【學位級別】：博士
【學位授予年份】：2008
【分類號】：R329.2

【參考文獻】

相關(guān)期刊論文 前10條

1 張豐;李青;秦緒軍;王文勇;張麗英;趙一嶺;洪柳;聶蕾;;細胞密度與細胞增殖關(guān)系的初步分析[J];醫(yī)學研究生學報;2006年01期

2 王軍升,周宏灝;CYP3A基因表達與調(diào)控的分子機制[J];生理科學進展;1998年02期

3 江青艷,張守全,傅偉龍;微重力組織工程的產(chǎn)生與發(fā)展[J];中國生物工程雜志;2002年03期

4 胡還章;徐小平;高毅;楊繼震;;生物人工肝治療豬實驗性急性肝衰[J];世界華人消化雜志;2001年02期

5 楊繼震;生物人工肝的研究現(xiàn)狀[J];世界華人消化雜志;1999年03期

6 徐小平,高毅,楊繼震;人工肝生物材料人肝細胞系CL-1的微載體培養(yǎng)[J];世界華人消化雜志;1999年03期

7 陳柯,高毅,潘玉先,楊繼震;正常原代豬肝細胞方瓶微載體靜止培養(yǎng)[J];世界華人消化雜志;1999年03期

8 張桂蓉,羅永艾,梅同華,周向東;非小細胞肺癌組織中血管內(nèi)皮生長因子、CD44v6及Ki67的表達水平及與患者預后的關(guān)系[J];中華結(jié)核和呼吸雜志;2003年07期

9 戴軍,陸倫根,曾民德,李繼強,華靜,茅益民,范竹萍,彭延申,邱德凱;肝細胞色素P450 2E1在實驗性肝纖維化組織中的表達[J];肝臟;2000年01期

10 Evangelos Dalakas;;Prevalence of porcine endogenous retrovirus in Chinese pig breeds and in patients treated with a porcine liver cell-based bioreactor[J];World Journal of Gastroenterology;2005年30期

，

本文編號：1695594