電刺激對H9C2心肌肌母細胞分化的影響

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許立翔(Li-Xiang Xu) 查詢紙本館藏

畢業系所

化學工程與材料工程學系

論文名稱

電刺激對H9C2心肌肌母細胞分化的影響
(The effects of electrical stimulation on the differentiation of H9C2 cardiomyoblast)

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摘要(中)

由於聚吡咯(polypyrrole, PPy)具有良好光學性質及生物相容性，因此我們以 PPy 製作成電刺激裝置，將大鼠心肌肌母細胞 H9C2 培養於裝置上並於兩側連接電極，再接上 Arduino 控制器與電源供應器，利用定電壓(constant DC)與單相電脈衝(pulse, 1Hz, 2 ms)以高與低電場(7、1V/cm)，探討不同參數的電刺激對 H9C2 細胞活性、排列與分化的影響。電脈衝在高低電場都能提高細胞活性的表現，但定電壓則要在較高電壓時才能提高細胞活性。兩種通電模式在高電場的刺激下都能促進細胞排列，定電壓組因其持續通電的特性能使排列更為明顯。就 qPCR 分析來看，高電場的電刺激能在早期增進 GATA4 心臟轉錄因子上調控。cTnT 及 α-actinin 作為心臟分化的後期指標，α-actinin 不論基因或蛋白質表現主要均由電場強度決定，與電刺激型式無關。 qPCR 結果顯示電壓增加有助於 cTnT 的基因表現，其中電脈衝較定電壓對 cTnT 上調控效果更佳，但是西方點墨分析結果顯示 cTnT 蛋白質表現與只跟電壓有關，推測可能是高電場電脈衝影響細胞活性所致。最後在免疫螢光染色得知，因為高電場的刺激促進細胞排列，也因此促進了成熟心肌細胞的順向排列，這些結果顯示以聚吡咯基材為媒介的電流刺激可促進心肌分化，極具心臟組織工程的潛力。

摘要(英)

Polypyrrole (PPy) exhibits good optical property and biocompatibility, so we used PPy to construct a bioreactor for electrical stimulation application. Rat cardiomyoblast cells, H9C2, were seeded to the bioreactor which was connected with electrodes in two opposite ends. We applied external power supply and Arduino controller to generate monophasic pulse current (1 Hz and 2 ms) and constant direct current (DC) in electric fields of 1 or 7 V/cm to investigate the effects of electrical stimulation mode as well as electrical fields on the activity, morphology and differentiation of H9C2 cells. The MTT results showed that pulse stimulation at 1 and 7 V/cm both promoted cell activity, whereas constant DC demonstrated cell activity improvement only at 7 V/cm. Only 7 V/cm of pulse or constant DC can align H9C2 cells to parallel to electrical field, and constant DC resulted in better alignment than that of pulse group. The qPCR results showed that pulse and constant at 7 V/cm both enhanced the upregulation of GATA4, a cardiac transcription maker, in early stage of differentiation. Two mature cardiac markers, cardiac muscle troponin T (cTnT) and α-actinin, were also investigated during differentiation. The qPCR and Western blotting results showed that the RNA and protein expression levels of α-actinin were mainly dependent on electric field and independent of stimulation mode. Both RNA and protein expression levels of cTnT also increased with electric fields. However, the qPCR results indicated that pulse stimulation up-regulated cTnT gene higher than that of the constant DC group, especially at 7 V/cm. Interestingly, the Western blotting results showed that these two stimulation modes demonstrated similar cTnT protein expression. We deduced that pulse stimulation decreased cell activity at 7 V/cm, and thus reduced cTnT synthesis. Finally, the immunostaining results demonstrated that 7 V/cm of pulse or constant DC both can align differentiated H9C2 cells due to their effects on cell orientation. Overall, our results suggested that PPy mediated electrical stimulation can promote cardiac myogenesis, and is potential for cardiac tissue engineering application
(DC) in electric fields of 1 or 7 V/cm to investigate the effects of
electrical stimulation mode as well as electrical fields on the activity,
morphology and differentiation of H9C2 cells. The MTT results showed
that pulse stimulation at 1 and 7 V/cm both promoted cell activity,
whereas constant DC demonstrated cell activity improvement only at 7
V/cm. Only 7 V/cm of pulse or constant DC can align H9C2 cells to
parallel to electrical field, and constant DC resulted in better alignment
than that of pulse group. The qPCR results showed that pulse and
constant at 7 V/cm both enhanced the upregulation of GATA4, a cardiac
transcription maker, in early stage of differentiation. Two mature cardiac
markers, cardiac muscle troponin T (cTnT) and α-actinin, were also
investigated during differentiation. The qPCR and Western blotting
results showed that the RNA and protein expression levels of α-actinin
were mainly dependent on electric field and independent of stimulation
mode. Both RNA and protein expression levels of cTnT also increased
with electric fields. However, the qPCR results indicated that pulse
stimulation up-regulated cTnT gene higher than that of the constant DC
group, especially at 7 V/cm. Interestingly, the Western blotting results
showed that these two stimulation modes demonstrated similar cTnT
protein expression. We deduced that pulse stimulation decreased cell
activity at 7 V/cm, and thus reduced cTnT synthesis. Finally, the
immunostaining results demonstrated that 7 V/cm of pulse or constant
DC both can align differentiated H9C2 cells due to their effects on cell
orientation. Overall, our results suggested that PPy mediated electrical
stimulation can promote cardiac myogenesis, and is potential for cardiac
tissue engineering application

關鍵字(中)

★ 心肌細胞
★ H9C2
★ 分化
★ 電刺激

關鍵字(英)

★ cardiomyoblast
★ H9C2
★ electrical stimulation
★ differentiation

論文目次

目錄
摘要.................................................................................................................................i
圖目錄...........................................................................................................................vi
表目錄........................................................................................................................ viii
第一章緒論..................................................................................................................1
1-1 前言.....................................................................................................................1
第二章文獻回顧..........................................................................................................4
2-1 心肌.....................................................................................................................4
2-1-1 心肌分化......................................................................................................6
2-1-2 心肌肌母細胞 H9C2...................................................................................8
2-2 組織工程.............................................................................................................9
2-2-1 心肌組織工程............................................................................................10
2-3 電刺激對心肌的影響.......................................................................................12
2-3-1 電刺激裝置................................................................................................14
2-3-2 電刺激促進心肌分化................................................................................19
2-3-3 電刺激促進心肌排列................................................................................23
2-3-4 電刺激的參數............................................................................................28
2-4 與心臟相關的基因...........................................................................................30
2-4-1 基因對心肌的影響....................................................................................31
2-4-2 電刺激對基因的影響................................................................................33
第三章實驗藥品、儀器及方法................................................................................36
3-1 實驗藥品...........................................................................................................36
3-1-1 材料製備藥品............................................................................................36
3-1-2 生物實驗藥品............................................................................................37
3-2 實驗儀器...........................................................................................................44
3-3 實驗方法...........................................................................................................47
3-3-1 設計、組裝生物刺激裝置........................................................................47
3-3-2 細胞培養、繼代、冷凍及解凍................................................................49
v
3-3-3 分化血清配製及分化................................................................................52
3-3-4 控制區的配置、程式................................................................................53
3-3-5 脈衝訊號的量測........................................................................................54
3-3-6 電刺激對心肌肌管分化的影響................................................................56
3-3-7 物理刺激對生物活性的影響....................................................................59
3-3-8 即時聚合酶反應儀 (Real-time PCR).......................................................61
3-3-9 cTnT/α-actinin 免疫螢光染色分析 ...........................................................65
3-3-10 細胞排列分析..........................................................................................67
3-3-11 西方點墨 (Western blot) ........................................................................68
第四章結果與討論....................................................................................................77
4-1 電刺激對 H9C2 肌母細胞的影響...................................................................77
4-1-1 MTT 生物活性分析...................................................................................77
4-1-2 細胞排列分析............................................................................................81
4-1-3 基因 qPCR 分析........................................................................................86
4-1-4 西方點墨(Western Blot)分析....................................................................91
4-1-5 免疫螢光染色(IF)分析 .............................................................................95
結論............................................................................................................................103
參考文獻....................................................................................................................105

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指導教授

胡威文(Wei-Wen Hu)

審核日期

2022-4-6

推文