【摘要】：轉(zhuǎn)移是腫瘤高致死率及低存活率的首要誘因,而腫瘤轉(zhuǎn)移與腫瘤細(xì)胞的定向遷移能力緊密相關(guān)。內(nèi)源性生物電場(chǎng)廣泛存在于生物體內(nèi),大量研究結(jié)果暗示了腫瘤轉(zhuǎn)移與內(nèi)源性生物電場(chǎng)異變存在相關(guān)性。細(xì)胞普遍具有趨電性是導(dǎo)致該相關(guān)性的生理基礎(chǔ)之一,且電信號(hào)是指導(dǎo)細(xì)胞定向遷移的主導(dǎo)信號(hào)。肺癌目前是全球癌癥死亡主因,而中國(guó)是世界第一肺癌大國(guó),云南省肺癌發(fā)病率則居于中國(guó)首位�；诖�,本論文選取了PG49、A549、Calu-3三種不同來(lái)源的肺腺癌細(xì)胞為研究對(duì)象,以支氣管上皮細(xì)胞HBE為對(duì)照,基于Time lapse技術(shù),通過(guò)施加外源性微直流電場(chǎng)量化分析了4種細(xì)胞對(duì)電場(chǎng)的響應(yīng)模式,并對(duì)比了4種細(xì)胞趨電性的差異。同時(shí)深入探討了細(xì)胞集群模式、營(yíng)養(yǎng)狀態(tài)以及偽足對(duì)肺腺癌細(xì)胞趨電性的影響。通過(guò)上述實(shí)驗(yàn),以期為全面揭示腫瘤轉(zhuǎn)移機(jī)制提供參考數(shù)據(jù),并為腫瘤電診療技術(shù)的發(fā)展和應(yīng)用提供理論基礎(chǔ)。實(shí)驗(yàn)結(jié)果如下:1)肺腺癌細(xì)胞PG49、A549、Calu-3及支氣管上皮細(xì)胞HBE均具有趨電性本實(shí)驗(yàn)將四種細(xì)胞暴露于強(qiáng)度為0、2、4、6 v/cm的電場(chǎng)中,依次量化分析了每種細(xì)胞對(duì)電場(chǎng)的響應(yīng)模式,并對(duì)比了3種肺腺癌細(xì)胞與支氣管上皮細(xì)胞趨電性的差異。結(jié)果表明:加入電場(chǎng)后,四種細(xì)胞從隨機(jī)遷移模式改變?yōu)槌螂妶?chǎng)負(fù)極定向遷移,其中HBE、Calu-3、PG49在電場(chǎng)強(qiáng)度為4v/cm時(shí)方向性指數(shù)最大,而A549在6v/cm時(shí)最大。HBE和Calu-3細(xì)胞在電場(chǎng)中持續(xù)朝向負(fù)極遷移的穩(wěn)定性高于無(wú)電場(chǎng)作用的對(duì)照組,且在電壓強(qiáng)度為4v/cm時(shí),HBE和Calu-3的方向持續(xù)性指數(shù)均最大;A549和PG49細(xì)胞在電場(chǎng)中的方向持續(xù)性指數(shù)在電壓強(qiáng)度為2v/cm時(shí),方向持續(xù)性指數(shù)最大。此外,電場(chǎng)亦可顯著加快細(xì)胞的遷移速度,Calu-3和PG49分別在電場(chǎng)強(qiáng)度2v/cm和4v/cm時(shí)遷移速度達(dá)到最大值,而HBE和A549均在電場(chǎng)強(qiáng)度6v/cm時(shí)遷移速度最快。雖然三種肺腺癌細(xì)胞與正常體細(xì)胞HBE的趨電方向一致,但肺腺癌細(xì)胞遷移的軌跡速度和位移速度顯著快于正常體細(xì)胞。該結(jié)果暗示趨電性在肺腺癌細(xì)胞系中廣泛存在,且其更加快速的遷移速度可能是其具有異常增強(qiáng)的轉(zhuǎn)移能力的基礎(chǔ)。2)Calu-3單細(xì)胞及群體細(xì)胞均具有趨電性,但趨電模式具有差異本實(shí)驗(yàn)以無(wú)電場(chǎng)作用的Calu-3細(xì)胞為對(duì)照組,以暴露于電場(chǎng)強(qiáng)度為2v/cm、4v/cm和6v/cm的Calu-3細(xì)胞為實(shí)驗(yàn)組,研究了外源微直流電場(chǎng)對(duì)人肺腺癌細(xì)胞Calu-3遷移的影響。結(jié)果表明Calu-3細(xì)胞可通過(guò)單細(xì)胞和群體細(xì)胞兩種形式遷移,直流電場(chǎng)對(duì)這兩種遷移形式具有顯著的影響:對(duì)照組單細(xì)胞和群體性的Calu-3隨機(jī)遷移。施加直流電場(chǎng)后,單細(xì)胞和群體性的Calu-3朝向負(fù)極定向遷移,趨電能力顯著高于對(duì)照組。電場(chǎng)強(qiáng)度為4v/cm時(shí),單細(xì)胞和群體性的Calu-3方向性最明顯,遷移方向最恒定,遷移速度最快。相同強(qiáng)度的電場(chǎng)作用下,Calu-3群體細(xì)胞遷移的方向性指數(shù)和方向持續(xù)性均高于單細(xì)胞遷移,但單細(xì)胞遷移的軌跡速度和位移速度快于群體細(xì)胞遷移,表明群體細(xì)胞遷移并非單細(xì)胞遷移的簡(jiǎn)單聚合,二者具有本質(zhì)差別。3)缺少血清促使A549趨電性增強(qiáng)本實(shí)驗(yàn)通過(guò)施加0、2、4、6v/cm電場(chǎng)于含有血清和未加血清的A549細(xì)胞,探究?jī)煞N情況下A549細(xì)胞趨電性遷移特征。結(jié)果顯示:施加電場(chǎng)后,有血清和無(wú)血清的A549細(xì)胞均朝向電場(chǎng)負(fù)極遷移,隨電壓強(qiáng)度增大,兩者的趨電能力逐漸增強(qiáng)。在不同電場(chǎng)強(qiáng)度中無(wú)血清A549的方向性指數(shù)及方向穩(wěn)定性均高于加血清A549。在電場(chǎng)強(qiáng)度為6v/cm時(shí),兩者的方向性指數(shù)最大。并且電場(chǎng)對(duì)有血清和無(wú)血清A549的遷移速度均有促進(jìn)作用,但無(wú)血清A549在電場(chǎng)中的遷移速度快于有血清A549,電場(chǎng)強(qiáng)度為6v/cm時(shí),兩者的速度均為最大。上述結(jié)果說(shuō)明,缺失血清并不會(huì)導(dǎo)致細(xì)胞喪失趨電能力,反而促進(jìn)細(xì)胞在電場(chǎng)中的遷移。4)電場(chǎng)影響了A549細(xì)胞的偽足性狀本實(shí)驗(yàn)對(duì)比了在強(qiáng)度分別為0 v/cm和6v/cm電場(chǎng)作用下A549細(xì)胞偽足性狀的差異。結(jié)果表明:在兩種不同強(qiáng)度電場(chǎng)作用下,A549偽足的形成數(shù)量和形成速度無(wú)顯著差異。然而,電場(chǎng)指導(dǎo)偽足集中朝向電場(chǎng)負(fù)極方向延伸,并顯著提高了A549細(xì)胞裂生型偽足的占比。施加外源電場(chǎng)后,靠近負(fù)極一側(cè)的偽足形成速度、數(shù)量均高于朝正極一側(cè)及無(wú)電場(chǎng)作用時(shí)的正極與負(fù)極;在細(xì)胞靠近負(fù)極一側(cè)的裂生型偽足顯著多于靠近正極一側(cè)的裂生型偽足數(shù)量。此結(jié)果符合偽足中心模型的預(yù)測(cè),即電場(chǎng)并不刺激細(xì)胞產(chǎn)生更多的偽足,但是可以在偽足形成的固有周期的基礎(chǔ)上,調(diào)控偽足的形成方式、分布區(qū)域、延伸方向來(lái)保證細(xì)胞對(duì)電場(chǎng)方向的敏感性。
[Abstract]:The metastasis is the primary cause of high mortality and low survival rate of the tumor, and the tumor metastasis is closely related to the directional migration ability of the tumor cells. The endogenous bioelectric field is widely present in the organism, and a large number of research results suggest that the tumor metastasis is related to the abnormal change of the endogenous bioelectric field. The general trend of the cell is the physiological basis that leads to the correlation, and the electrical signal is the dominant signal that directs the cell to directionally migrate. Lung cancer is currently the main cause of global cancer death, and China is the first major lung cancer country in the world, and the incidence of lung cancer in Yunnan Province is the first in China. Based on this, three different kinds of lung adenocarcinoma cells from PG49, A549 and Calu-3 were selected as the research object, and the response pattern of 4 cells to the electric field was analyzed by applying exogenous micro-DC field. The difference of the four types of cells was compared. At the same time, the effects of cell cluster model, nutritional status and pseudo-foot on the chemotaxis of lung adenocarcinoma were discussed. The above-mentioned experiment is to provide the reference data for the comprehensive disclosure of the tumor metastasis mechanism and to provide a theoretical basis for the development and application of the tumor electrotherapy technology. The results of the experiment are as follows:1) The lung adenocarcinoma cells PG49, A549, Calu-3 and the bronchial epithelial cell HBE have an electric field in which four cells are exposed to an electric field with the intensity of 0,2,4 and 6 v/ cm, and the response pattern of each cell to the electric field is quantitatively analyzed, The difference of the electrical properties between the three lung adenocarcinoma cells and the bronchial epithelial cells was compared. The results showed that after the electric field was added, the four cells were changed from the random migration mode to the negative directional migration of the electric field, where the directivity index of the HBE, Calu-3 and PG49 was the largest when the electric field intensity was 4 v/ cm, while the A549 was the largest at 6 v/ cm. The stability of HBE and Calu-3 cells in the electric field is higher than that of the control group with no electric field effect, and the direction persistence index of both the HBE and the Calu-3 is the largest when the voltage intensity is 4 v/ cm, and the direction persistence index of the A549 and PG49 cells in the electric field is 2 v/ cm. The direction persistence index is the largest. In addition, the electric field can significantly increase the migration speed of the cells, and the migration speed of Calu-3 and PG49 at the electric field intensity of 2 v/ cm and 4 v/ cm reaches the maximum value, while both the HBE and the A549 have the fastest migration speed when the electric field intensity is 6 v/ cm. Although the three lung adenocarcinoma cells were in the same direction as the normal somatic cell HBE, the speed and speed of the migration of the lung adenocarcinoma cells were significantly faster than that of the normal cells. The results suggest that the chemotaxis are widely present in the lung adenocarcinoma cell line, and the more rapid migration speed may be the basis of the transfer ability with abnormal enhancement. In this experiment, the Calu-3 cells with no electric field effect were used as the control group, and the Calu-3 cells with an electric field intensity of 2 v/ cm,4 v/ cm and 6 v/ cm were taken as the experimental group, and the effect of the external micro-direct current field on the calu-3 migration of human lung adenocarcinoma cells was studied. The results show that the calu-3 cells can be transferred through two forms of single cell and group cells, and the direct current field has a significant effect on the two types of migration: control group single cell and group-based Calu-3 random migration. When the DC field was applied, the cell and group Calu-3 migrated toward the negative electrode, and the electrokinetic ability was significantly higher than that of the control group. When the intensity of the electric field is 4v/ cm, the directivity of the calu-3 of the single cell and the group is the most obvious, the migration direction is the most constant, and the migration speed is the fastest. Under the action of the same intensity of electric field, the directional index and the direction persistence of the cell migration of the Calu-3 group were higher than that of the single cell, but the track velocity and the displacement speed of the single cell migration were faster than that of the group cells, indicating that the migration of the population cells was not a simple polymerization of single cell migration. The electric field of A549 cells was determined by applying 0,2,4,6 v/ cm electric field to A549 cells containing serum and serum without serum. The results showed that both serum and serum-free A549 cells were migrated towards the negative electrode of the electric field after the electric field was applied. The directional index and the direction stability of the serum-free A549 in different electric field intensities were higher than that of the serum A549. When the electric field intensity is 6 v/ cm, the directivity index of the two is the largest. And the electric field can promote the migration speed of the serum and the serum-free A549, but the migration speed of the serum-free A549 in the electric field is faster than that of the serum A549, and when the electric field intensity is 6 v/ cm, the speed of the two is the maximum. The results indicated that the absence of serum did not lead to the loss of electrical ability of the cells, but rather the migration of the cells in the electric field.4) The electric field affected the pseudo-foot character of the A549 cells. This experiment compared the difference of the pseudo-foot characters of the A549 cells under the action of 0 v/ cm and 6 v/ cm, respectively. The results showed that there was no significant difference in the number of formation and the formation speed of A549 pseudo-foot under the action of two different intensity electric fields. However, that electric field guide the pseudo-foot set to extend in the negative direction of the electric field, and significantly improve the proportion of the open-type pseudo-foot of the A549 cell. When a foreign electric field is applied, the pseudo-foot formation speed near the negative electrode side is higher than that of the positive electrode and the negative electrode when the positive electrode side and the non-electric field effect are applied, and the crack-type pseudo-foot on the side of the cell near the negative electrode is significantly more than the number of the split-type pseudo-feet near the positive electrode side. The result is in accordance with the prediction of the pseudo-foot center model, that is, the electric field does not stimulate the cells to generate more pseudo-foot, but the formation mode, the distribution region and the extension direction of the pseudo-foot can be controlled on the basis of the inherent period of the pseudo-foot formation, so that the sensitivity of the cells to the direction of the electric field can be ensured.
【學(xué)位授予單位】：云南師范大學(xué)
【學(xué)位級(jí)別】：碩士
【學(xué)位授予年份】：2017
【分類號(hào)】：R734.2

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