發(fā)布時間：2019-04-11 13:02

【摘要】：轉移是腫瘤高致死率及低存活率的首要誘因,而腫瘤轉移與腫瘤細胞的定向遷移能力緊密相關。內源性生物電場廣泛存在于生物體內,大量研究結果暗示了腫瘤轉移與內源性生物電場異變存在相關性。細胞普遍具有趨電性是導致該相關性的生理基礎之一,且電信號是指導細胞定向遷移的主導信號。肺癌目前是全球癌癥死亡主因,而中國是世界第一肺癌大國,云南省肺癌發(fā)病率則居于中國首位。基于此,本論文選取了PG49、A549、Calu-3三種不同來源的肺腺癌細胞為研究對象,以支氣管上皮細胞HBE為對照,基于Time lapse技術,通過施加外源性微直流電場量化分析了4種細胞對電場的響應模式,并對比了4種細胞趨電性的差異。同時深入探討了細胞集群模式、營養(yǎng)狀態(tài)以及偽足對肺腺癌細胞趨電性的影響。通過上述實驗,以期為全面揭示腫瘤轉移機制提供參考數據,并為腫瘤電診療技術的發(fā)展和應用提供理論基礎。實驗結果如下:1)肺腺癌細胞PG49、A549、Calu-3及支氣管上皮細胞HBE均具有趨電性本實驗將四種細胞暴露于強度為0、2、4、6 v/cm的電場中,依次量化分析了每種細胞對電場的響應模式,并對比了3種肺腺癌細胞與支氣管上皮細胞趨電性的差異。結果表明:加入電場后,四種細胞從隨機遷移模式改變?yōu)槌螂妶鲐摌O定向遷移,其中HBE、Calu-3、PG49在電場強度為4v/cm時方向性指數最大,而A549在6v/cm時最大。HBE和Calu-3細胞在電場中持續(xù)朝向負極遷移的穩(wěn)定性高于無電場作用的對照組,且在電壓強度為4v/cm時,HBE和Calu-3的方向持續(xù)性指數均最大;A549和PG49細胞在電場中的方向持續(xù)性指數在電壓強度為2v/cm時,方向持續(xù)性指數最大。此外,電場亦可顯著加快細胞的遷移速度,Calu-3和PG49分別在電場強度2v/cm和4v/cm時遷移速度達到最大值,而HBE和A549均在電場強度6v/cm時遷移速度最快。雖然三種肺腺癌細胞與正常體細胞HBE的趨電方向一致,但肺腺癌細胞遷移的軌跡速度和位移速度顯著快于正常體細胞。該結果暗示趨電性在肺腺癌細胞系中廣泛存在,且其更加快速的遷移速度可能是其具有異常增強的轉移能力的基礎。2)Calu-3單細胞及群體細胞均具有趨電性,但趨電模式具有差異本實驗以無電場作用的Calu-3細胞為對照組,以暴露于電場強度為2v/cm、4v/cm和6v/cm的Calu-3細胞為實驗組,研究了外源微直流電場對人肺腺癌細胞Calu-3遷移的影響。結果表明Calu-3細胞可通過單細胞和群體細胞兩種形式遷移,直流電場對這兩種遷移形式具有顯著的影響:對照組單細胞和群體性的Calu-3隨機遷移。施加直流電場后,單細胞和群體性的Calu-3朝向負極定向遷移,趨電能力顯著高于對照組。電場強度為4v/cm時,單細胞和群體性的Calu-3方向性最明顯,遷移方向最恒定,遷移速度最快。相同強度的電場作用下,Calu-3群體細胞遷移的方向性指數和方向持續(xù)性均高于單細胞遷移,但單細胞遷移的軌跡速度和位移速度快于群體細胞遷移,表明群體細胞遷移并非單細胞遷移的簡單聚合,二者具有本質差別。3)缺少血清促使A549趨電性增強本實驗通過施加0、2、4、6v/cm電場于含有血清和未加血清的A549細胞,探究兩種情況下A549細胞趨電性遷移特征。結果顯示:施加電場后,有血清和無血清的A549細胞均朝向電場負極遷移,隨電壓強度增大,兩者的趨電能力逐漸增強。在不同電場強度中無血清A549的方向性指數及方向穩(wěn)定性均高于加血清A549。在電場強度為6v/cm時,兩者的方向性指數最大。并且電場對有血清和無血清A549的遷移速度均有促進作用,但無血清A549在電場中的遷移速度快于有血清A549,電場強度為6v/cm時,兩者的速度均為最大。上述結果說明,缺失血清并不會導致細胞喪失趨電能力,反而促進細胞在電場中的遷移。4)電場影響了A549細胞的偽足性狀本實驗對比了在強度分別為0 v/cm和6v/cm電場作用下A549細胞偽足性狀的差異。結果表明:在兩種不同強度電場作用下,A549偽足的形成數量和形成速度無顯著差異。然而,電場指導偽足集中朝向電場負極方向延伸,并顯著提高了A549細胞裂生型偽足的占比。施加外源電場后,靠近負極一側的偽足形成速度、數量均高于朝正極一側及無電場作用時的正極與負極;在細胞靠近負極一側的裂生型偽足顯著多于靠近正極一側的裂生型偽足數量。此結果符合偽足中心模型的預測,即電場并不刺激細胞產生更多的偽足,但是可以在偽足形成的固有周期的基礎上,調控偽足的形成方式、分布區(qū)域、延伸方向來保證細胞對電場方向的敏感性。
[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.
【學位授予單位】：云南師范大學
【學位級別】：碩士
【學位授予年份】：2017
【分類號】：R734.2

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