wnt-3a上调CCN1表达促进终末期肾病骨骼肌衰老(硕士)

wnt-3a上调CCN1表达促进终末期肾病骨骼肌衰老(硕士)(论文28000字)
中文摘要
目的：
1．    探究终末期肾病患者血清CCN1表达水平与肌少症的相关性。
2．    研究wnt-3a对骨骼肌细胞增殖和衰老的作用
3．    验证wnt-3a是否通过上调CCN1的表达而促进骨骼肌细胞衰老。
方法：
一、临床资料分析
分析2018年6月至12月在XX医院收住的≥65周岁尚未行肾脏替代治疗的终末期肾病患者55例及同期进行体检的年龄、高血压、糖尿病等系统性疾病史相匹配的非CKD、≥65周岁的老年人110例和20-30周岁成年人20例的血清标本，收集终末期肾病患者和老年人的基本资料，包括年龄，性别，身高，体重；相关临床资料，包括全身骨骼肌质量指数和握力，血肌酐，血清总蛋白，白蛋白，总胆固醇，甘油三酯，HDL-胆固醇，LDL-胆固醇，尿酸，尿比重，24h尿蛋白；既往史如高血压，糖尿病，手术史和肿瘤史。采用人CCN1 ELISA试剂盒检测血清CCN1表达量。
二、体外实验验证
以小鼠骨骼肌细胞株（C2C12）为研究对象
1.    重组Wnt-3a细胞毒性检测：不同剂量小鼠重组 wnt-3a（0、100、500、1000、1500ng/ml）分别处理C2C12骨骼肌细胞8h、12h和24h后，检测细胞活率。
2.    不同剂量小鼠重组 wnt-3a（0、100、500、1000ng/ml）处理C2C12骨骼肌细胞12h后，qRT-PCR法检测CCN1、细胞衰老指标p53和p16、细胞增殖指标MyOD及整合素受体α1、α2、α3、αv、α6、β1、β2、β3的mRNA表达量，western blot法检测CCN1、p53、pRB和p16蛋白表达量。
3.    Wnt信号通路阻断剂DKK-1阻断wnt-3a，DKK-1与wnt-3a共同作用12h后，qRT-PCR法检测CCN1、p53、p16和MyOD mRNA表达量，western blot法检测CCN1、p53、pRB和p16蛋白表达量。
4.    尿毒症小鼠血清刺激：取野生型、假手术和尿毒症小鼠模型的血清，用DMEM培养液配置成含10%相应组别血清的培养液，分别处理C2C12细胞12h，加等量不含血清的DMEM培养液作为空白对照组。qRT-PCR法检测CCN1、p53、p16和MyOD mRNA表达量。

结果：
1.    55例终末期肾病患者中符合肌少症诊断者24例（占43.6%），与无肌少症组比较显示低BMI（t=7.238，P＜0.0001），低白蛋白（t=1.778，P=0.081），低握力（t=4.041，P=0.0002），低骨骼肌质量指数（t=2.263，P=0.028）为肌少症可能相关因素。
2.    110例老年人中符合肌少症诊断者45例（占40.9%），与无肌少症组组间比较显示低BMI（t=10.57，P＜0.0001），低甘油三酯（t=2.768，P=0.007），低握力（t=7.876，P＜0.0001），低骨骼肌质量指数（t=9.034，P＜0.0001）为肌少症可能相关因素。
3.    与正常成年人相比，终末期肾病患者血清CCN1（96.35 ± 10.80 ng/ml）、老年人血清CCN1（83.48 ± 8.46 ng/ml）均显著升高（P＜0.05），其中，终末期肾病患者血清CCN1含量较正常成年人升高2.2倍，老年人血清CCN1含量升高1.9倍；与终末期肾病无肌少症组比较，终末期肾病伴肌少症患者血清CCN1（119.3 ± 18.17 ng/ml）明显升高；与老年人无肌少症组比较，老年人肌少症患者血清CCN1（104.0 ± 12.17 ng/ml）明显升高。
4.    在5组不同剂量的小鼠重组wnt-3a分别处理8、12和24h后，与对照组相比，当浓度低于1g/ml时，wnt-3a未引起C2C12细胞活率的显著变化。而当1.5g/ml 重组wnt-3a处理12和24h后，观察到C2C12细胞活率明显下降。
5.    在4组不同剂量的小鼠重组wnt-3a处理12h后，与空白对照组相比，CCN1 mRNA表达量明显升高，且呈剂量依赖性，随wnt-3a浓度升高而升高。
6.    1g/ml 重组wnt-3a处理C2C12细胞12h后，与对照组（0g/ml）相比，CCN1、P53和P16 mRNA表达量明显升高，MyOD mRNA表达量下降，整合素受体α6和β1 mRNA表达量明显升高。DKK-1抑制剂可明显抑制wnt-3a上调CCN1表达的作用，并可抑制wnt-3a诱导C2C12细胞衰老的作用，恢复骨骼肌细胞的增殖能力。
7.    在野生型、假手术和尿毒症小鼠血清条件下生长的C2C12细胞，与野生型血清组相比，尿毒症血清条件下CCN1，p53和p16 mRNA表达量升高，MyOD mRNA表达量下降。
结论：
1.    老年人肌少症和终末期肾病肌少症患者，均伴有低BMI、低肌肉质量分数和低握力，老年人肌少症还伴有低甘油三酯。
2.    血清CCN1水平与肌少症相关，老年人肌少症和终末期肾病肌少症患者血清CCN1水平较成年人均明显升高，血清CCN1含量可能是肌少症的预测指标。
3.    在体外实验中，一定浓度的wnt-3a可上调C2C12细胞CCN1的表达，诱导细胞衰老过程，同时可抑制细胞增殖能力。
4.    当wnt-3a信号通路被DKK-1抑制后，wnt-3a诱导的CCN1在mRNA和蛋白水平的上调作用被阻断，被抑制的细胞增殖能力恢复，诱导的衰老指标p53和p16表达量逆转。
5.    在野生型、假手术和尿毒症小鼠血清条件下生长的C2C12细胞，尿毒症血清环境下CCN1表达升高，衰老指标p53和p16升高，细胞增殖指标MyOD下降，表明尿毒症血清体外仍可诱导C2C12骨骼肌细胞衰老，抑制细胞增殖能力。
关键词：CCN1,肌少症，wnt-3a，衰老，终末期肾病，C2C12。

Wnt-3a induces CCN1 expression leading to skeletal muscle senescence in end-stage renal disease
Abstract
Objective:
1.    To investigate the relationship between serum CCN1 expression and sarcopenia in patients with end-stage renal disease.
2.    To explore the effect of wnt-3a on skeletal muscle cell proliferation and senescence.
3.    To verify whether wnt-3a induces skeletal muscle cell senescence through stimulates CCN1 expression.
Method:
1.    Clinical data analysis
From June to December 2018, 55 patients with end-stage renal disease who had not undergone renal replacement therapy at the first hospital affiliated to Wenzhou Medical University were analyzed, 110 cases of non-CKD and ≥65 years old were selected as the elderly group, and 20 normal adults aged 20-30 years old were selected as the control group. These people were matched 1:2 by age, systemic diseases such as hypertension and diabetes. Collected their serum samples and basic data including age, gender, height, weight; relevant clinical data, including whole body skeletal muscle mass index and grip strength, serum creatinine, serum total protein, albumin, total cholesterol, triglycerides, HDL-cholesterol, LDL-cholesterol, uric acid, specific gravity of urine, 24-hour urine protein; past medical history, including hypertension, diabetes, surgical history and tumor history. Serum CCN1 expression was measured using the human CCN1 ELISA kit.
2.    In vitro experiments
a) Recombinant Wnt-3a cytotoxicity assay: Cell viability was measured after treatment of C2C12 skeletal muscle cells with different doses of recombinant wnt-3a (0, 100, 500, 1000, 1500 ng/ml) for 8 hours, 12 hours and 24 hours, respectively.
b) Different doses of recombinant wnt-3a (0, 100, 500, 1000 ng/ml) were used to treat C2C12 cells for 12 hours. The mRNA expression levels of CCN1, senescence indicators p53 and p16, proliferation index MyOD and integrin receptors α1, α2, α3, αv, α6, β1, β2, β3 were assessed by qRT-PCR. The expression of CCN1, p53 and p16 protein were assessed by Western blot.
c) Wnt signaling inhibitors DKK-1: DKK-1 and wnt-3a together treated C2C12 cells for 12 hours. The mRNA expression levels of CCN1, p53, p16, MyOD and integrin receptors were assessed by qRT-PCR. The expression of CCN1, p53 and p16 protein were assessed by Western blot.
d) Treated with serum of uremic model mice: C2C12 myoblasts were grown for 12 hours in DMEM medium containing 10% wild-type, sham-operated and uremic models mice serum, respectively. Serum-free DMEM medium was used as control group. The expression levels of CCN1, p53, p16 and MyOD mRNA were detected by qRT-PCR.
Result:
1. Among the 55 patients with uremia, 24 patients (43.6%) were diagnosed with sarcopenia. Compared with the non-sarcopenia group, low BMI (t=7.238, P<0.0001), low albumin (t=1.778, P=0.081), low grip strength (t=4.041, P=0.0002) and low skeletal muscle mass index (t=2.263, P=0.028) were possible factors associated with sarcopenia.
2. Among the 110 elderly humans, 45 (40.9%) were diagnosed with sarcopenia. The comparison between the groups indicated that the low BMI (t=10.57, P<0.0001), low triglyceride (t=2.768, P=0.007), low grip strength (t=7.876, P<0.0001) and low skeletal muscle mass index (t=9.034, P<0.0001) were possible factors associated with sarcopenia.
3. CCN1 was increased in serum from patients with uremia (96.35 ± 10.80 ng/ml) and elderly humans (83.48 ± 8.46 ng/ml) compared with young control. The level of CCN1 in serum from patients with end-stage renal disease was 2.2 times higher than that of normal adults, and the level of CCN1 in serum from the elderly humans was increased by 1.9 times; Compared with the uremic non-sarcopenia patients, CCN1 was significantly elevated in serum from patients with uremia and sarcopenia (119.3 ± 18.17 ng/ml); Compared with the elderly non-sarcopenia humans, CCN1 was significantly elevated in serum from elderly patients with sarcopenia (104.0 ± 12.17 ng/ml).
4. C2C12 cells were treated with different dosages of mouse recombinant wnt-3a for 8 hours, 12 hours and 24 hours respectively, compared with the control group, wnt-3a did not cause significant changes of C2C12 cell viability at the concentrations lower than 1g/ml. However, significantly decreased cell viability was observed after 1.5g/ml recombinant wnt-3a treatment.
5. After treatment with different dosages of recombinant wnt-3a for 12 hours, the expression of CCN1 mRNA was significantly increased in a dose-dependent manner, which increased with the increase of wnt-3a concentration.
6. After treated with 1ug/ml recombinant Wnt-3a for 12 hours, compared with the control group (0g/ml), the expression of CCN1, P53 and P16 mRNA were significantly increased, and the expression of MyOD mRNA was decreased, the integrin receptor α6 and β1 were significantly increased. This effect was blocked by DKK-1, the addition of DKK-1 significantly inhibited the effect of wnt-3a on up-regulating CCN1 expression and cellular senescence. In addition, the proliferation of C2C12 cells was restored.
7. C2C12 cells grown under serum conditions of wild-type, sham-operated and uremic mice, respectively. qRT-PCR revealed that the mRNAs of CCN1, P53 and P16 were increased in uremia serum condition compared with wild-type serum. MyOD mRNA expression was decreased in cells treated with serum from uremic mice.
Conclusion:
1. Elderly humans with sarcopenia and uremia patients with sarcopenia were associated with low BMI, low muscle mass index and low grip strength. Elderly humans with sarcopenia were also associated with low triglycerides.
2. Serum CCN1 levels were associated with sarcopenia. Serum CCN1 levels in elderly and uremia patients with sarcopenia were significantly higher than normal adults. Elevated serum CCN1 levels may be predictors of sarcopenia.
3. In vitro, a certain concentration of wnt-3a can induce the expression of CCN1 to promote muscle cellular senescence and attenuate cell proliferation.
4. When the wnt-3a signaling pathway was inhibited by DKK-1, the up-regulation of CCN1 induced by wnt-3a at mRNA and protein levels were blocked, and the proliferation of muscle cell was restored, the levels of cell senescence indicators P53 and P16 were also reversed.
5. The mRNA expression of P53 and P16 were increased and the MyOD were decreased in the serum of uremia, indicating that uremia serum stimulated the senescence process of C2C12 cells and inhibited cell proliferation.
Keywords: CCN1, sarcopenia, wnt-3a, senescence, end-stage renal disease, C2C12.

目录
缩略词表••••••••••••••••••••••••••••••••••••••••••••••••• 1
中文摘要••••••••••••••••••••••••••••••••••••••••••••••••• 3
英文摘要••••••••••••••••••••••••••••••••••••••••••••••••• 6
引言•••••••••••••••••••••••••••••••••••••••••••••••••••••9
材料与方法•••••••••••••••••••••••••••••••••••••••••••••••11
结果•••••••••••••••••••••••••••••••••••••••••••••••••••••25
分析与讨论•••••••••••••••••••••••••••••••••••••••••••••••34
参考文献•••••••••••••••••••••••••••••••••••••••••••••••••37
致谢•••••••••••••••••••••••••••••••••••••••••••••••••••••40
综述及参考文献•••••••••••••••••••••••••••••••••••••••••••41
 
缩略词表
缩略词    英文全称    中文全称
AP    Ammonium persulfate    过硫酸铵
BCA    Bicinchoninic acid    二辛可酸
BMI    Body mass index    体重指数
BSA    Bovine serum albumin    牛血清白蛋白
Cyr61    Cysteine-rich 61    富含半胱氨酸61
CCK-8    Cell Counting Kit-8    细胞活率测定盒
CKD    Chronic kidney disease    慢性肾脏病
DEPC    Diethypyrocarbonate    焦碳酸二乙酯
DMEM    Dulbecco's Modified Eagle Medium    Dulbecco改良的培养液
DMSO    Dimethyl sulfoxide    二甲基亚砜
DNA    Desoxyribonucleic acid    脱氧核糖核酸
DKK-1    Dickkopf-1    Dickkopf-1
ECM    Extracellular matrix    细胞外基质
EDTA    Ethylene diamine tetraacetic acid    乙二胺四乙酸
ELISA    Enzyme-linked immunosorbent assay    酶联免疫吸附测定
IGF    Insulin-like growth factor    胰岛素样生长因子
MAPK    Mitogen-activated protein kinases    丝裂原激活蛋白激酶
MMP    Matrix metalloproteinases    基质金属蛋白酶
PAGE    Polyacrylamide gel electrophoroesis    聚丙烯酰胺凝胶电泳
PBS    Phosphate buffered saline    磷酸盐缓冲液
PDGF    Platelet-derived growth factor    血小板衍生生长因子
PMSF    Phenylmethanesulfonylfluoride    本甲基磺酰氟
PVDF    Polyvinylidene difluoride    聚偏二氟乙烯
RB    Retinoblastoma protein     视网膜母细胞瘤蛋白
RCC    Renal cell carcinoma    肾细胞癌
RIPA    Radio Immunoprecipitation Assay    放射免疫沉淀法
ROS    Reactive oxygen species    活性氧
qRT-PCR    Quantitative real-time polymerase chain reaction    实时定量聚合酶链式反应
Sasp    Senescence-associated secretory phenotype    衰老相关分泌表型
SDS    Sodium dodecyl sulfate    十二烷基磺酸钠
TBS     Triethanolamine buffered saline    三乙醇胺缓冲盐水溶液
TEMED    Tetramethylethylenediamine    四甲基乙二胺
TGF-β    Transforming growth factor-β    转化生长因子-β
Tris    Trishydroxymethylaminomethane    三羟甲基氨基甲烷
VEGF    Vascular endothelial growth factor    血管内皮生长因子
Wnt-3a    Wingless/int1-3a    无翅家族蛋白-3a
YAP    Yes-associated protein    Yes-相关蛋白