乙型肝炎病毒包膜蛋白诱导肝癌细胞系内质网应激的研究

doi:10.3877/cma.j.issn.1674-1358.2016.03.027

目的

探讨乙型肝炎病毒前-S缺失和野生型外膜大蛋白（LHBs）诱导内质网应激反应。

方法

运用脂质体转染技术将pcDNA3.1-S、pcDNA3.1-L、pcDNA3.1-L△30和pcDNA3.1-L△182等真核表达质粒转染至Huh7细胞，并应用qRT-PCR和Western blot方法检测转染细胞前-S1、前-S2、HBsAg和GRP78的mRNA和蛋白表达水平。

结果

转染HBV包膜蛋白各组的HBsAg蛋白相对表达量（分别为0.92、0.83、0.91、1.75、2.53和2.06）高于空白对照（0.00）和pcDNA3.1（+）转染组（0.00）（F = 247.38、P = 0.000）；转染HBV包膜蛋白各组的GRP78的蛋白相对表达量（分别为1.4、1.47、1.55、1.75、1.8和1.9）高于空白对照（1.18）和pcDNA3.1(+)转染组（1.23）（F = 11.623、P = 0.000）；且GRP78蛋白相对表达量与前-S1、前-S2抗原和HBsAg表达量呈正相关（相关系数r分别为0.884、0.728和0.816）。

结论

前-S缺失型/野生型LHBs及HBsAg均能诱导Huh7细胞发生内质网应激反应，提示其可能参与肝癌发生。

关键词: 肝炎病毒, 乙型, 外膜大蛋白, 表面抗原, 葡萄糖调节蛋白78, 内质网应激

Objective

To explore endoplasmic reticulum stress in Huh7 cells induced by wild type and pre-S mutant envelope proteins of HBV.

Methods

The recombinant eukaryotic expression vectors of pcDNA3.1-S, pcDNA3.1-L, pcDNA3.1-L△30 and pcDNA3.1-L△182 were transfected into Huh7 cells through liposome transfection, then the protein exrpression of pre-S1, pre-S2, HBsAg and Grp78 were detected by Western blot, and Grp78 mRNA transcripts were detected by qRT-PCR.

Results

The relative expression of HBsAg protein in Huh7 cells transfected with recombinant eukaryotic expression vectors (0.92, 0.83, 0.91, 1.75, 2.53 and 2.06, respectively) were significantly higher than that in blank control cells (0.00) and that in cells transfected with pcDNA3.1(+) (0.00) (F = 247.38, P = 0.000). The relative expression of GRP78 protein in Huh7 cells transfected with recombinant eukaryotic expression vectors (1.4, 1.47, 1.55, 1.75, 1.8 and 1.9, respectively) were significantly higher than that in blank control cells (1.18) and that in cells transfected with pcDNA3.1(+) (1.23) (F = 11.623, P = 0.000). The relative expression of GRP78 protein had opositive correlation with that of pre-S1, pre-S2 and HBsAg (r = 0.884, 0.728 and 0.816, respectively).

Conclusions

Expression of envelope proteins of HBV could induce endoplasmic reticulum stress, indicating they might involve in hepatocarcinogenesis.

Key words: Hepatitis B virus, Hepatitis B virus large surface protein, Hepatitis B virus surface antigen, Glucose regulated protein 78, Endoplasmic reticulum stress

表1 RT-PCR所有引物序列、位置和长度

名称	序列（5′→3′）	位置	长度（bp）
18srRNA-F	CCTGGATACCGCAGCTAGGA	846～861	108
18srRNA-R	GCGGCGCAATACGAATGCCCC	953～932	108
GRP78-F	CCCTGTCTTCTCAGCATCAA	1 154～1 173	101
GRP78-R	TAGACCGGAACAGATCCATGT	1 273～1 253	101
HBsAg-F	TGCCCGTTTGTCCTCTACTT	466～485	101
HBsAg-R	CCGAAGGTTTTGTACAGCAA	584～565	101
LHBs-F	ATGGGAGGTTGGTCTTCC	2 848～2 865	116
LHBs-R	TTTGAGTTGGCTCCGAAC	2 963～2 946	116

表1 RT-PCR所有引物序列、位置和长度

名称	序列（5′→3′）	位置	长度（bp）
18srRNA-F	CCTGGATACCGCAGCTAGGA	846～861	108
18srRNA-R	GCGGCGCAATACGAATGCCCC	953～932	108
GRP78-F	CCCTGTCTTCTCAGCATCAA	1 154～1 173	101
GRP78-R	TAGACCGGAACAGATCCATGT	1 273～1 253	101
HBsAg-F	TGCCCGTTTGTCCTCTACTT	466～485	101
HBsAg-R	CCGAAGGTTTTGTACAGCAA	584～565	101
LHBs-F	ATGGGAGGTTGGTCTTCC	2 848～2 865	116
LHBs-R	TTTGAGTTGGCTCCGAAC	2 963～2 946	116

表2 GRP78、HBsAg和LHBs等mRNA相对表达量（±s）

分组	GRP78	HBsAg	LHBs
空白组	1.00 ± 0.12	1.00 ± 0.14	1.00 ± 0.17
pcDNA3.1	1.03 ± 0.18	2.40 ± 0.13	1.11 ± 0.18
pcDNA3.1-S	1.23 ± 0.28	16 561.36 ± 1 490.21 ^b	1.22 ± 0.21
pcDNA3.1-L	1.42 ± 0.22^a	22 847.78 ± 1 515.87^b	26.51 ± 1.68^b
pcDNA3.1-L△30	1.33 ± 0.21	11 877.42 ± 770.54^b	21.11 ± 2.37^b
pcDNA3.1-S + pcDNA3.1-L	1.37 ± 0.25	37 047.28 ± 1 680.17^b	23.24 ± 2.07^b
pcDNA3.1-S + pcDNA3.1-L△30	1.28 ± 0.27	31 560.34 ± 1 824.43^b	18.27 ± 1.17^b
pcDNA3.1-S + pcDNA3.1-L△183	1.43 ± 0.31	43 271.72 ± 2 863.84^{b *}	20.37 ± 1.85^b

表2 GRP78、HBsAg和LHBs等mRNA相对表达量（±s）

分组	GRP78	HBsAg	LHBs
空白组	1.00 ± 0.12	1.00 ± 0.14	1.00 ± 0.17
pcDNA3.1	1.03 ± 0.18	2.40 ± 0.13	1.11 ± 0.18
pcDNA3.1-S	1.23 ± 0.28	16 561.36 ± 1 490.21 ^b	1.22 ± 0.21
pcDNA3.1-L	1.42 ± 0.22^a	22 847.78 ± 1 515.87^b	26.51 ± 1.68^b
pcDNA3.1-L△30	1.33 ± 0.21	11 877.42 ± 770.54^b	21.11 ± 2.37^b
pcDNA3.1-S + pcDNA3.1-L	1.37 ± 0.25	37 047.28 ± 1 680.17^b	23.24 ± 2.07^b
pcDNA3.1-S + pcDNA3.1-L△30	1.28 ± 0.27	31 560.34 ± 1 824.43^b	18.27 ± 1.17^b
pcDNA3.1-S + pcDNA3.1-L△183	1.43 ± 0.31	43 271.72 ± 2 863.84^{b *}	20.37 ± 1.85^b

图1 HBsAg和LHBs mRNA相对表达量

图2 转染细胞Grp78、HBsAg、pre-S1和pre-S2等蛋白表达

图3 GRP78蛋白相对表达量与前-S1、前-S2抗原和HBsAg表达的相关性

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Endoplasmic reticulum stress in Huh7 cell induced by envelope proteins of hepatitis B virus

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Endoplasmic reticulum stress in Huh7 cell induced by envelope proteins of hepatitis B virus