三种不同来源肺腺癌细胞的趋电性研究
发布时间：2019-04-11 13:02
【摘要】：转移是肿瘤高致死率及低存活率的首要诱因,而肿瘤转移与肿瘤细胞的定向迁移能力紧密相关。内源性生物电场广泛存在于生物体内,大量研究结果暗示了肿瘤转移与内源性生物电场异变存在相关性。细胞普遍具有趋电性是导致该相关性的生理基础之一,且电信号是指导细胞定向迁移的主导信号。肺癌目前是全球癌症死亡主因,而中国是世界第一肺癌大国,云南省肺癌发病率则居于中国首位。基于此,本论文选取了PG49、A549、Calu-3三种不同来源的肺腺癌细胞为研究对象,以支气管上皮细胞HBE为对照,基于Time lapse技术,通过施加外源性微直流电场量化分析了4种细胞对电场的响应模式,并对比了4种细胞趋电性的差异。同时深入探讨了细胞集群模式、营养状态以及伪足对肺腺癌细胞趋电性的影响。通过上述实验,以期为全面揭示肿瘤转移机制提供参考数据,并为肿瘤电诊疗技术的发展和应用提供理论基础。实验结果如下:1)肺腺癌细胞PG49、A549、Calu-3及支气管上皮细胞HBE均具有趋电性本实验将四种细胞暴露于强度为0、2、4、6 v/cm的电场中,依次量化分析了每种细胞对电场的响应模式,并对比了3种肺腺癌细胞与支气管上皮细胞趋电性的差异。结果表明:加入电场后,四种细胞从随机迁移模式改变为朝向电场负极定向迁移,其中HBE、Calu-3、PG49在电场强度为4v/cm时方向性指数最大,而A549在6v/cm时最大。HBE和Calu-3细胞在电场中持续朝向负极迁移的稳定性高于无电场作用的对照组,且在电压强度为4v/cm时,HBE和Calu-3的方向持续性指数均最大;A549和PG49细胞在电场中的方向持续性指数在电压强度为2v/cm时,方向持续性指数最大。此外,电场亦可显著加快细胞的迁移速度,Calu-3和PG49分别在电场强度2v/cm和4v/cm时迁移速度达到最大值,而HBE和A549均在电场强度6v/cm时迁移速度最快。虽然三种肺腺癌细胞与正常体细胞HBE的趋电方向一致,但肺腺癌细胞迁移的轨迹速度和位移速度显著快于正常体细胞。该结果暗示趋电性在肺腺癌细胞系中广泛存在,且其更加快速的迁移速度可能是其具有异常增强的转移能力的基础。2)Calu-3单细胞及群体细胞均具有趋电性,但趋电模式具有差异本实验以无电场作用的Calu-3细胞为对照组,以暴露于电场强度为2v/cm、4v/cm和6v/cm的Calu-3细胞为实验组,研究了外源微直流电场对人肺腺癌细胞Calu-3迁移的影响。结果表明Calu-3细胞可通过单细胞和群体细胞两种形式迁移,直流电场对这两种迁移形式具有显著的影响:对照组单细胞和群体性的Calu-3随机迁移。施加直流电场后,单细胞和群体性的Calu-3朝向负极定向迁移,趋电能力显著高于对照组。电场强度为4v/cm时,单细胞和群体性的Calu-3方向性最明显,迁移方向最恒定,迁移速度最快。相同强度的电场作用下,Calu-3群体细胞迁移的方向性指数和方向持续性均高于单细胞迁移,但单细胞迁移的轨迹速度和位移速度快于群体细胞迁移,表明群体细胞迁移并非单细胞迁移的简单聚合,二者具有本质差别。3)缺少血清促使A549趋电性增强本实验通过施加0、2、4、6v/cm电场于含有血清和未加血清的A549细胞,探究两种情况下A549细胞趋电性迁移特征。结果显示:施加电场后,有血清和无血清的A549细胞均朝向电场负极迁移,随电压强度增大,两者的趋电能力逐渐增强。在不同电场强度中无血清A549的方向性指数及方向稳定性均高于加血清A549。在电场强度为6v/cm时,两者的方向性指数最大。并且电场对有血清和无血清A549的迁移速度均有促进作用,但无血清A549在电场中的迁移速度快于有血清A549,电场强度为6v/cm时,两者的速度均为最大。上述结果说明,缺失血清并不会导致细胞丧失趋电能力,反而促进细胞在电场中的迁移。4)电场影响了A549细胞的伪足性状本实验对比了在强度分别为0 v/cm和6v/cm电场作用下A549细胞伪足性状的差异。结果表明:在两种不同强度电场作用下,A549伪足的形成数量和形成速度无显著差异。然而,电场指导伪足集中朝向电场负极方向延伸,并显著提高了A549细胞裂生型伪足的占比。施加外源电场后,靠近负极一侧的伪足形成速度、数量均高于朝正极一侧及无电场作用时的正极与负极;在细胞靠近负极一侧的裂生型伪足显著多于靠近正极一侧的裂生型伪足数量。此结果符合伪足中心模型的预测,即电场并不刺激细胞产生更多的伪足,但是可以在伪足形成的固有周期的基础上,调控伪足的形成方式、分布区域、延伸方向来保证细胞对电场方向的敏感性。
[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.
【学位授予单位】：云南师范大学
【学位级别】：硕士
【学位授予年份】：2017
【分类号】：R734.2
[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.
【学位授予单位】：云南师范大学
【学位级别】：硕士
【学位授予年份】：2017
【分类号】：R734.2
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