酪丝亮肽在人肝癌BEL-7402细胞中的定位及其跨膜转运机制的初步研究
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摘要
目的：
研究抗肿瘤三肽化合物酪丝亮肽(YSL)在肝癌细胞BEL-7402中的定位,并初步探讨小肽转运蛋白(PEPT1)在YSL跨膜转运中的作用。
方法：
1.建立荧光素5(6)-羧四甲基罗丹明琥珀酰亚氨酯标记YSL的方法,应用非变性聚丙烯酰胺凝胶电泳对荧光标记YSL进行纯化。
2.应用毛细管电泳技术和荧光分光光度法对荧光标记YSL进行纯度和稳定性鉴定；应用速率比色法测定人肝癌BEL-7402细胞受损后释放的乳酸脱氢酶来鉴定标记YSL的生物学活性。
3.以激光扫描共聚焦显微镜观察标记YSL在体外培养的人肝癌BEL-7402细胞中的分布以及是否与肿瘤细胞的线粒体存在共定位。
4.应用荧光偏振法测定YSL体外对人肝癌BEL-7402细胞膜流动性的影响,以流式细胞仪测定YSL体外对人肝癌BEL-7402细胞膜电位的影响,为进一步判定YSL进入肿瘤细胞的方式提供证据。
5.应用分光光度法测定YSL对分离的人肝癌BEL-7402细胞线粒体膜电位和线粒体肿胀的影响,观察YSL是否对肿瘤细胞的线粒体有直接作用。
6.应用免疫荧光方法,观察小肽转运蛋白PEPT1在人肝癌BEL-7402细胞的定位和表达。
7.应用MTS法测定PEPT1的底物GLY-GLN、GLY-SARCOSINE和GLY-GLY-GLY对YSL抑制人肝癌BEL-7402细胞增殖作用的影响；以倒置荧光显微镜观察三种PEPT1的底物对标记YSL进入BEL-7402细胞数量的影响,探讨YSL进入肿瘤细胞过程是否与PEPT1的功能存在关系。
8.应用实时荧光定量PCR法观察YSL对人肝癌细胞BEL-7402中PEPT1的mRNA水平的影响。
结果：
1.荧光物质5(6)-羧四甲基罗丹明琥珀酰亚氨酯可以稳定地标记YSL。经过20%的非变性聚丙烯酰胺凝胶电泳纯化后可以得到成分单一的荧光标记YSL。
2.合成的荧光标记YSL性质稳定：在-20℃条件下避光保存两周,YSL和5(6)羧四甲基罗丹明琥珀酰亚胺酯结合稳定未发生解离,标记YSL的荧光强度和荧光光谱也未发生明显的变化。并且荧光标记YSL体外损伤人肝癌BEL-7402细胞使之释放的LDH与标记前相比未发生明显的下降(P>0.05),即标记前后YSL的抗肿瘤生物学活性未发生明显改变。
3.荧光标记YSL在体外与人肝癌BEL-7402细胞共同孵育时能够进入细胞内,并且在细胞中呈聚集分布,与等浓度的5 (6) TAMRA SE在细胞的分布有明显的差别；标记YSL主要分布于BEL-7402细胞的细胞浆中,并且与肿瘤细胞的线粒体呈共定位。
4. YSL(1.6mg/ml)在体外作用BEL-7402细胞能够使细胞膜的微黏度增加,降低细胞膜的流动性；还可以降低肿瘤细胞膜的膜电位,造成细胞膜去极化。
5.YSL直接作用于体外分离的人肝癌BEL-7402细胞线粒体后,能够降低线粒体膜电位,影响线粒体膜通透性,造成线粒体肿胀。
6.在人肝癌BEL-7402细胞中检测到PEPT1蛋白的表达,PEPT1蛋白在BEL-7402细胞主要分布在细胞的胞膜和胞浆。
7. PEPT1的作用底物GLY-SARCOSINE、GLY-GLN和GLY-GLY-GLY可以减弱YSL对人肝癌BEL-7402细胞增殖的抑制作用,降低标记YSL在体外进入人肝癌BEL-7402细胞的数量。
8. YSL (1.6mg/ml)能够在体外上调人肝癌BEL-7402细胞中PEPT1 mRNA水平。
结论：
应用荧光物质5,6羧四甲基罗丹明琥珀酰亚氨酯可以稳定地标记YSL,并保持YSL原有的抗肿瘤生物活性。YSL可能是跨膜进入到肿瘤细胞的胞质内发挥抗肿瘤作用的,YSL进入肿瘤细胞的过程可能与肿瘤细胞中的小肽转运蛋白PEPT1的转运功能有关。进入肿瘤细胞胞质内的YSL发挥抗肿瘤作用的方式之一可能是通过损伤肿瘤细胞线粒体实现的。
Objective:
To explore the location of antitumor tripeptide compound tyroserleutide (YSL) in the human hepatocellular carcinoma BEL-7402 cells and its preliminary mechanism of transport mediated by human proton dependent peptide transporter.
Methods:
1. Establishing a synthetic method of fluorescent analogue of YSL and purified it by native PAGE.
2. The purity and the stability of the fluorescent analogue of YSL were assessed by capillary electrophoresis and fluorescent spectrophotometric assay; The biochemical activity of the fluorescent analogue of YSL was evaluated through the excretion of LDH of the injured human hepatocellular carcinoma BEL-7402 cells by speptrophotometric assay.
3. The subcellular distribution of the fluorescent analogue of YSL in the human hepatocellular carcinoma BEL-7402 cells and whether the fluorescent analogue of YSL were colocalized with the mitochondria of the human hepatocellular carcinoma BEL-7402 cells were observe by laser scanning confocal microscopy.
4. The effect of YSL on the membrane fluidity and plasma membrane potential of human hepatocellular carcinoma BEL-7402 cells in vitro were assessed by fluorescent polarization assay and flow cytometry to provide the evidence for the style of transporting into the tumor cells.
5. The effect of YSL on the mitochondrial membrane potential and mitochondrial swelling of human hepatocellular carcinoma BEL-7402 cells were measured by spectrophotometric assay to observe whether YSL has the direct effect on the mitochondria of the tumor cells.
6. The expression and the subcellular location of PEPT1 in the human hepatocellular carcinoma BEL-7402 cells were observed by fluorescent immunoassay.
7. The influence of GLY-GLN, GLY-SARCOSINE and GLY-GLY-GLY on the effect of the proliferative inhibition rate of YSL to the human hepatocellular carcinoma BEL-7402 cells was assessed by MTS method; The influence of GLY-GLN, GLY-SARCOSINE and GLY-GLY-GLY on the the YSL uptake of the human hepatocellular carcinoma BEL-7402 cells was observed by inverted fluorescent microscopy.
8. The effect of YSL on PEPT1 mRNA of human hepatocellular carcinoma BEL-7402 cells was observed by Real-time quantitative PCR.
Results:
1. The fluorescent material 5(6)-TAMRASE can conjugate with YSL stably. The fluorescent analogue of YSL can be purified by 20% native PAGE.
2. The purified fluorescent analogue of YSL was stable. There was no separation between the 5(6)-TAMRASE and YSL when it was stored at-20℃avoiding from light for 2 weeks and both of the fluorescent intensities and the spectrum had no great changes taken place. There was no significant decrease of the activity of LDH excreted by human hepatocelluar carcinoma BEL-7402 cells between the tumor cells labeled YSL treated and unlabeled YSL treated (P>0.05). The biological activity of the YSL had no obviously changes labeled before and after.
3. YSL fluorescent analogue can enter the human hepatocellular carcinoma BEL-7402 cells when they were incubated with the cells and distributed in the cells intensely. The distribution pattern of fluorescent analogue of YSL was different from that of the same doze of fluorescent material 5(6)-TAMRASE. YSL fluorescent analogue mainly located at the cytoplasm of the human heaptocarcinoma BEL-7402 cells and colocalization with the mitochondria.
4. YSL (1.6mg/ml) can decrease the membrane microviscosity and increase the membrane fluidity of the human heaptocellular carcinoma BEL-7402 cells in vitro. YSL can decrease the plasma membrane potential of the human heaptocellular carcinoma BEL-7402 cells in vitro and lead to the depolarizing of the cells.
5. YSL can decrease the isolated mitochondrial potential of the human heaptocellular carcinoma BEL-7402 cells, influence the permeability of the mitchondria and caused the mitochondrial swelling when it was incubated with the isolated mitochondria of the tumor cells in vitro.
6. The PEPT1 protein can express in the human heaptocellular carcinoma BEL-7402 cells and mainly located at the membrane and cytoplasm of the tumor cells.
7. The substrate of PEPT1 GLY-SARCOSINE,GLY-GLN and GLY-GLY-GLY can weaken the inhibition effect of YSL on human hepatocellular carcinoma BEL-7402 cells in vitro and inhibited the uptake of YSL by human hepatocellular carcinoma BEL-7402 cells in vitro respectively.
8. YSL (1.6mg/ml) can upregulate the PEPT1 rnRNA expression of the human hepatocellular carcinoma BEL-7402 cells in vitro.
Conclusion:
YSL can be labeled with fluorescent material 5(6)-TAMRASE stably and the antitumor biological activity of YSL was matained without any changes. We speculate that YSL can enter the human hepatocellular carcinoma BEL-7402 cells and located at the plasma to kill the tumor cells. PEPT1 in the human hepatocellular carcinoma BEL-7402 cells maybe involved in the transport process of YSL across the BEL-7402 cells membrane. One of the antitumor mechanisms of YSL when they were transported into the tumor cells was realized by injuring the mitochondria of the tumor cells.
研究抗肿瘤三肽化合物酪丝亮肽(YSL)在肝癌细胞BEL-7402中的定位,并初步探讨小肽转运蛋白(PEPT1)在YSL跨膜转运中的作用。
方法：
1.建立荧光素5(6)-羧四甲基罗丹明琥珀酰亚氨酯标记YSL的方法,应用非变性聚丙烯酰胺凝胶电泳对荧光标记YSL进行纯化。
2.应用毛细管电泳技术和荧光分光光度法对荧光标记YSL进行纯度和稳定性鉴定；应用速率比色法测定人肝癌BEL-7402细胞受损后释放的乳酸脱氢酶来鉴定标记YSL的生物学活性。
3.以激光扫描共聚焦显微镜观察标记YSL在体外培养的人肝癌BEL-7402细胞中的分布以及是否与肿瘤细胞的线粒体存在共定位。
4.应用荧光偏振法测定YSL体外对人肝癌BEL-7402细胞膜流动性的影响,以流式细胞仪测定YSL体外对人肝癌BEL-7402细胞膜电位的影响,为进一步判定YSL进入肿瘤细胞的方式提供证据。
5.应用分光光度法测定YSL对分离的人肝癌BEL-7402细胞线粒体膜电位和线粒体肿胀的影响,观察YSL是否对肿瘤细胞的线粒体有直接作用。
6.应用免疫荧光方法,观察小肽转运蛋白PEPT1在人肝癌BEL-7402细胞的定位和表达。
7.应用MTS法测定PEPT1的底物GLY-GLN、GLY-SARCOSINE和GLY-GLY-GLY对YSL抑制人肝癌BEL-7402细胞增殖作用的影响；以倒置荧光显微镜观察三种PEPT1的底物对标记YSL进入BEL-7402细胞数量的影响,探讨YSL进入肿瘤细胞过程是否与PEPT1的功能存在关系。
8.应用实时荧光定量PCR法观察YSL对人肝癌细胞BEL-7402中PEPT1的mRNA水平的影响。
结果：
1.荧光物质5(6)-羧四甲基罗丹明琥珀酰亚氨酯可以稳定地标记YSL。经过20%的非变性聚丙烯酰胺凝胶电泳纯化后可以得到成分单一的荧光标记YSL。
2.合成的荧光标记YSL性质稳定：在-20℃条件下避光保存两周,YSL和5(6)羧四甲基罗丹明琥珀酰亚胺酯结合稳定未发生解离,标记YSL的荧光强度和荧光光谱也未发生明显的变化。并且荧光标记YSL体外损伤人肝癌BEL-7402细胞使之释放的LDH与标记前相比未发生明显的下降(P>0.05),即标记前后YSL的抗肿瘤生物学活性未发生明显改变。
3.荧光标记YSL在体外与人肝癌BEL-7402细胞共同孵育时能够进入细胞内,并且在细胞中呈聚集分布,与等浓度的5 (6) TAMRA SE在细胞的分布有明显的差别；标记YSL主要分布于BEL-7402细胞的细胞浆中,并且与肿瘤细胞的线粒体呈共定位。
4. YSL(1.6mg/ml)在体外作用BEL-7402细胞能够使细胞膜的微黏度增加,降低细胞膜的流动性；还可以降低肿瘤细胞膜的膜电位,造成细胞膜去极化。
5.YSL直接作用于体外分离的人肝癌BEL-7402细胞线粒体后,能够降低线粒体膜电位,影响线粒体膜通透性,造成线粒体肿胀。
6.在人肝癌BEL-7402细胞中检测到PEPT1蛋白的表达,PEPT1蛋白在BEL-7402细胞主要分布在细胞的胞膜和胞浆。
7. PEPT1的作用底物GLY-SARCOSINE、GLY-GLN和GLY-GLY-GLY可以减弱YSL对人肝癌BEL-7402细胞增殖的抑制作用,降低标记YSL在体外进入人肝癌BEL-7402细胞的数量。
8. YSL (1.6mg/ml)能够在体外上调人肝癌BEL-7402细胞中PEPT1 mRNA水平。
结论：
应用荧光物质5,6羧四甲基罗丹明琥珀酰亚氨酯可以稳定地标记YSL,并保持YSL原有的抗肿瘤生物活性。YSL可能是跨膜进入到肿瘤细胞的胞质内发挥抗肿瘤作用的,YSL进入肿瘤细胞的过程可能与肿瘤细胞中的小肽转运蛋白PEPT1的转运功能有关。进入肿瘤细胞胞质内的YSL发挥抗肿瘤作用的方式之一可能是通过损伤肿瘤细胞线粒体实现的。
Objective:
To explore the location of antitumor tripeptide compound tyroserleutide (YSL) in the human hepatocellular carcinoma BEL-7402 cells and its preliminary mechanism of transport mediated by human proton dependent peptide transporter.
Methods:
1. Establishing a synthetic method of fluorescent analogue of YSL and purified it by native PAGE.
2. The purity and the stability of the fluorescent analogue of YSL were assessed by capillary electrophoresis and fluorescent spectrophotometric assay; The biochemical activity of the fluorescent analogue of YSL was evaluated through the excretion of LDH of the injured human hepatocellular carcinoma BEL-7402 cells by speptrophotometric assay.
3. The subcellular distribution of the fluorescent analogue of YSL in the human hepatocellular carcinoma BEL-7402 cells and whether the fluorescent analogue of YSL were colocalized with the mitochondria of the human hepatocellular carcinoma BEL-7402 cells were observe by laser scanning confocal microscopy.
4. The effect of YSL on the membrane fluidity and plasma membrane potential of human hepatocellular carcinoma BEL-7402 cells in vitro were assessed by fluorescent polarization assay and flow cytometry to provide the evidence for the style of transporting into the tumor cells.
5. The effect of YSL on the mitochondrial membrane potential and mitochondrial swelling of human hepatocellular carcinoma BEL-7402 cells were measured by spectrophotometric assay to observe whether YSL has the direct effect on the mitochondria of the tumor cells.
6. The expression and the subcellular location of PEPT1 in the human hepatocellular carcinoma BEL-7402 cells were observed by fluorescent immunoassay.
7. The influence of GLY-GLN, GLY-SARCOSINE and GLY-GLY-GLY on the effect of the proliferative inhibition rate of YSL to the human hepatocellular carcinoma BEL-7402 cells was assessed by MTS method; The influence of GLY-GLN, GLY-SARCOSINE and GLY-GLY-GLY on the the YSL uptake of the human hepatocellular carcinoma BEL-7402 cells was observed by inverted fluorescent microscopy.
8. The effect of YSL on PEPT1 mRNA of human hepatocellular carcinoma BEL-7402 cells was observed by Real-time quantitative PCR.
Results:
1. The fluorescent material 5(6)-TAMRASE can conjugate with YSL stably. The fluorescent analogue of YSL can be purified by 20% native PAGE.
2. The purified fluorescent analogue of YSL was stable. There was no separation between the 5(6)-TAMRASE and YSL when it was stored at-20℃avoiding from light for 2 weeks and both of the fluorescent intensities and the spectrum had no great changes taken place. There was no significant decrease of the activity of LDH excreted by human hepatocelluar carcinoma BEL-7402 cells between the tumor cells labeled YSL treated and unlabeled YSL treated (P>0.05). The biological activity of the YSL had no obviously changes labeled before and after.
3. YSL fluorescent analogue can enter the human hepatocellular carcinoma BEL-7402 cells when they were incubated with the cells and distributed in the cells intensely. The distribution pattern of fluorescent analogue of YSL was different from that of the same doze of fluorescent material 5(6)-TAMRASE. YSL fluorescent analogue mainly located at the cytoplasm of the human heaptocarcinoma BEL-7402 cells and colocalization with the mitochondria.
4. YSL (1.6mg/ml) can decrease the membrane microviscosity and increase the membrane fluidity of the human heaptocellular carcinoma BEL-7402 cells in vitro. YSL can decrease the plasma membrane potential of the human heaptocellular carcinoma BEL-7402 cells in vitro and lead to the depolarizing of the cells.
5. YSL can decrease the isolated mitochondrial potential of the human heaptocellular carcinoma BEL-7402 cells, influence the permeability of the mitchondria and caused the mitochondrial swelling when it was incubated with the isolated mitochondria of the tumor cells in vitro.
6. The PEPT1 protein can express in the human heaptocellular carcinoma BEL-7402 cells and mainly located at the membrane and cytoplasm of the tumor cells.
7. The substrate of PEPT1 GLY-SARCOSINE,GLY-GLN and GLY-GLY-GLY can weaken the inhibition effect of YSL on human hepatocellular carcinoma BEL-7402 cells in vitro and inhibited the uptake of YSL by human hepatocellular carcinoma BEL-7402 cells in vitro respectively.
8. YSL (1.6mg/ml) can upregulate the PEPT1 rnRNA expression of the human hepatocellular carcinoma BEL-7402 cells in vitro.
Conclusion:
YSL can be labeled with fluorescent material 5(6)-TAMRASE stably and the antitumor biological activity of YSL was matained without any changes. We speculate that YSL can enter the human hepatocellular carcinoma BEL-7402 cells and located at the plasma to kill the tumor cells. PEPT1 in the human hepatocellular carcinoma BEL-7402 cells maybe involved in the transport process of YSL across the BEL-7402 cells membrane. One of the antitumor mechanisms of YSL when they were transported into the tumor cells was realized by injuring the mitochondria of the tumor cells.
引文
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