光动力疗法联合NS-398抑制Bcl-2促进QBC939细胞凋亡的机制研究

姜海涛; 黄左安; 黄丹丹; 项健健; 汪午轲; 陈云杰

doi:10.16766/j.cnki.issn.1674-4152.001765

光动力疗法联合NS-398抑制Bcl-2促进QBC939细胞凋亡的机制研究

doi: 10.16766/j.cnki.issn.1674-4152.001765

姜海涛^{1, 2, 3},
黄左安^{2, 3, 4},
黄丹丹^{2, 3, 4},
项健健^{1, 2, 3},
汪午轲^{1, 2, 3},
陈云杰^{1, 2, 3, ,}

1.
中国科学院大学宁波华美医院外二科，浙江宁波 315010
2.
浙江省消化系统肿瘤诊治及研究重点实验室，浙江宁波 315010
3.
中国科学院大学宁波生命与健康产业研究院, 浙江宁波 315010
4.
中国科学院大学宁波华美医院医学实验部, 浙江宁波 315010

基金项目:

华美研究基金 2017HMKY11

华美研究基金 2019HMKY67

浙江省消化系统肿瘤诊治及研究重点实验室 2019E10020

宁波市消化系统肿瘤临床医学研究中心 2019A21003

详细信息

通讯作者:
陈云杰，E-mail：ybyfish@163.com

中图分类号: R735.8 R730.5
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- 被引次数: 0
出版历程
- 收稿日期: 2020-01-19
- 网络出版日期: 2022-02-19

The Mechanism study of the promotion of photodynamic therapy combined with NS-398 on the apoptosis of bile duct cancer cells QBC939 through inhibiting Bcl-2

JIANG Hai-tao^{1, 2, 3},
HUANG Zuo-an^{2, 3, 4},
HUANG Dan-dan^{2, 3, 4},
XIANG Jian-jian^{1, 2, 3},
WANG Wu-ke^{1, 2, 3},
CHEN Yun-jie^{1, 2, 3
, ,}

1.
Department of General Surgery, HwaMei Hospital, University of Chinese Academy of Sciences, Ningbo, Zhejiang 315010, China

摘要

摘要: 目的研究光动力疗法(PDT)和选择性环氧合酶-2(COX-2)抑制剂NS-398联合对人胆管癌细胞QBC939凋亡的影响及其机理。方法 QBC939细胞体外培养后分组：光动力组、NS-398组、联合实验组和空白对照组，分别将0、2、4、6、8、10 μg/mL浓度的血卟啉衍生物(HPD)在0、5、10、15 J/cm²的光照强度和0、25、50、100、200 μmol/L浓度的NS-398联合作用于QBC939细胞；采用细胞增殖实验(CCK8)检测生长抑制率(R)；流式细胞法检测细胞凋亡；荧光定量RT-PCR检测Bcl-2基因表达；免疫细胞化学测定细胞浆中Bcl-2表达；ELISA测定细胞上清中Bcl-2蛋白。结果 PDT和NS-398在体外均能抑制QBC939细胞生长，当HPD浓度8 μg/mL，光照强度5 J/cm²联合50 μmol/L的NS-398，R值约达95%，联合实验组与其他3组比较均有统计学差异(P < 0.05)，继续增加2种强度，R变化不明显；流式细胞法表明PDT和NS-398联合能促进QBC939细胞早期凋亡(F=3 224.753, P < 0.001)。荧光定量RT-PCR显示联合能抑制细胞中Bcl-2表达(F=6 262.227, P < 0.001)；SP免疫细胞化学显示联合能抑制细胞浆中Bcl-2表达(F=1 355.577, P < 0.001)；ELISA显示联合能抑制细胞上清中Bcl-2分泌(F=5 123.387, P < 0.001)。结论 PDT和NS-398联合可明显抑制QBC939细胞生长，诱导早期凋亡，对生长的抑制可能是通过抑制Bcl-2基因和蛋白的表达，促进其早期凋亡实现的。
- 光动力疗法 /
- 选择性环氧合酶-2抑制剂 /
- 胆管癌 /
- B淋巴细胞瘤-2 /
- 凋亡
Abstract: Objective To study the effect of photodynamic therapy (PDT) combined with NS-398 on apoptosis of cholangiocarcinoma QBC939 cells and mechanism. Methods QBC939 cells were cultured and divided into four groups, namely, the photodynamic group, NS-398 group, joint experimental group and blank control group, which were exposed to 0, 2, 4, 6, 8 and 10 μg/mL of hematoporphyrin derivatives (HPD) with 0, 5, 10 and 15 J/cm² light radiation and 0, 25, 50, 100 and 200 μmol/L NS-398, respectively. The growth inhibition ratio of the QBC939 cells (R) was measured and calculated by cell proliferation experiment (CCK8). Flow cytometry was used to measure cell apoptosis. The mRNA and protein expression levels of Bcl-2 were measured by real-time PCR, immunocytochemistry and ELISA. Results Both PDT and NS-398 could inhibit the growth of QBC939 cells in vitro. When HPD concentration was 8 μ g/ml, light intensity was 5 J/cm², and NS-398 was 50 μmol/L, the R value was about 95%. There were significant differences between the combined experimental group and other groups (P < 0.05). If light radiation and NS-398 were increased, then the result would show no statistical differences. PDT combined with NS-398 could promote QBC939 cell apoptosis at the early stage through flow cytometry (F=3 224.753, P < 0.001). Real-time PCR demonstrated it could suppress the expression of Bcl-2 gene (F=6 262.227, P < 0.001). SP immunocytochemistry showed it could inhibit the expression of Bcl-2 (F=1 355.577, P < 0.001). ELISA showed it could inhibit the secretion of Bcl-2 (F=5 123.387, P < 0.001). Conclusion PDT combined with NS-398 could suppress the growth and promote early apoptosis of QBC939 cells. The inhibitory effect may be achieved by inhibiting the expression of Bcl-2 gene and protein, thus promoting the early apoptosis.
- Photodynamic therapy /
- Selective cyclooxygenase-2 inhibitor /
- Bile duct cancer /
- B-cell lymphoma-2 /
- Apoptosis

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图 1 PDT和NS-398联合对QBC939细胞凋亡的影响

注：A为空白对照组；B为光动力组；C为NS-398组；D为联合实验组。

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图 2 QBC939细胞中Bcl-2基因的相对表达量

注：与空白对照组比较，^aP < 0.05；与光动力组比较，^bP < 0.05；与NS-398组比较，^cP < 0.05。

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图 3 Bcl-2免疫细胞化学结果(苏木素染色，×200)

注：A为联合实验组；B为光动力组；C为NS-398组；D为空白对照组。

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图 4 QBC939细胞上清中Bcl-2的分泌量

注：与空白对照组比较，^aP < 0.05；与光动力组比较，^bP < 0.05；与NS-398组比较，^cP < 0.05。

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表 1 引物序列与最佳退火温度

基因	引物	引物序列	退火温度(℃)
Bcl-2	上游	5'-GATTGTGGCCTTCTTTGAGTT-3'	54
	下游	5'-AGTTCCACAAAGGCATCCCA-3'
hGAPDH	上游	5'-AACAGCCTCAAGATCATCAGCAA-3'	54
	下游	5'-GACTGTGGTCATGAGTCCTTCCA-3'

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表 2 PDT和NS-398联合作用QBC939细胞的R值(x ±s，%)

NS-398(μmol/L)	光照(J/cm²)	HPD(mg/L)
NS-398(μmol/L)	光照(J/cm²)	0	2	4	6	8	10
0	0	0.00±0.00	1.21±0.08	5.26±0.70	7.12±0.19	10.17±0.89	10.02±0.84
	5	1.07±0.11	4.20±0.45	24.18±1.15	30.18±1.02	38.77±2.04	70.21±2.60
	10	1.48±0.09	5.18±0.63	31.45±2.02	34.09±2.15	40.22±2.05	75.18±2.07
	15	1.51±0.14	6.27±0.71	40.47±2.08	61.05±2.02	70.08±2.13	85.54±2.19
25	0	20.42±1.09	20.75±1.12	20.81±1.44	20.96±1.52	21.19±1.49	21.62±1.36
	5	21.55±1.13	25.42±1.51	55.58±1.57	60.15±1.30	62.57±1.11	66.10±1.25
	10	23.42±1.37	30.18±1.20	58.15±1.68	62.09±1.26	65.14±1.45	72.58±1.37
	15	25.60±1.16	32.19±1.18	60.10±1.65	64.19±1.32	79.20±1.06	90.09±1.22
50	0	48.37±2.04	48.58±2.11	48.79±2.03	48.95±2.14	49.12±2.02	49.32±2.01
	5	50.44±2.11	52.48±2.17	70.46±1.57	80.55±1.63^a	95.26±1.75^a	95.38±1.43^a
	10	52.12±2.57	55.12±2.26	72.38±1.45	84.47±1.62^a	95.31±1.17^a	95.49±1.38^a
	15	53.23±2.52	56.18±2.23	75.98±1.39	87.49±1.59^a	95.48±1.25^a	95.55±1.69^a
100	0	57.62±2.51	57.74±2.36	57.83±2.29	57.98±2.17	58.22±2.21	58.45±2.39
	5	58.24±2.45	59.26±2.12	71.65±1.74	85.17±1.40^a	95.42±1.43^a	95.46±1.18^a
	10	59.76±2.24	60.86±1.75	75.40±1.81	87.56±1.61^a	95.47±1.20^a	95.53±1.11^a
	15	60.39±2.53	63.14±2.10	78.30±1.45	90.02±1.57^a	95.57±1.31^a	95.58±1.35^a
200	0	65.27±2.84	65.44±2.32	65.56±2.17	65.72±2.31	65.86±2.15	65.97±2.13
	5	66.34±2.11	67.13±2.18	80.35±1.50	90.15±1.44^a	95.46±1.31^a	95.53±1.17^a
	10	68.29±2.65	68.46±2.04	82.25±1.39	92.01±1.39^a	95.51±1.22^a	95.62±1.13^a
	15	69.33±2.61	70.10±2.19	85.37±1.51	93.55±1.23^a	95.63±1.10^a	95.68±1.19^a
注：与NS-398单一因素和光动力单一因素组相比，^aP＜0.05。

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