wnt-3a上調CCN1表達促進終末期腎病骨骼肌衰老(碩士)

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wnt-3a上調CCN1表達促進終末期腎病骨骼肌衰老(碩士)(論文28000字)
中文摘要
目的:
1.    探究終末期腎病患者血清CCN1表達水平與肌少癥的相關性。
2.    研究wnt-3a對骨骼肌細胞增殖和衰老的作用
3.    驗證wnt-3a是否通過上調CCN1的表達而促進骨骼肌細胞衰老。
方法:
一、臨床資料分析
分析2018年6月至12月在XX醫院收住的≥65周歲尚未行腎臟替代治療的終末期腎病患者55例及同期進行體檢的年齡、高血壓、糖尿病等系統性疾病史相匹配的非CKD、≥65周歲的老年人110例和20-30周歲成年人20例的血清標本,收集終末期腎病患者和老年人的基本資料,包括年齡,性別,身高,體重;相關臨床資料,包括全身骨骼肌質量指數和握力,血肌酐,血清總蛋白,白蛋白,總膽固醇,甘油三酯,HDL-膽固醇,LDL-膽固醇,尿酸,尿比重,24h尿蛋白;既往史如高血壓,糖尿病,手術史和腫瘤史。采用人CCN1 ELISA試劑盒檢測血清CCN1表達量。
二、體外實驗驗證
以小鼠骨骼肌細胞株(C2C12)為研究對象
1.    重組Wnt-3a細胞毒性檢測:不同劑量小鼠重組 wnt-3a(0、100、500、1000、1500ng/ml)分別處理C2C12骨骼肌細胞8h、12h和24h后,檢測細胞活率。
2.    不同劑量小鼠重組 wnt-3a(0、100、500、1000ng/ml)處理C2C12骨骼肌細胞12h后,qRT-PCR法檢測CCN1、細胞衰老指標p53和p16、細胞增殖指標MyOD及整合素受體α1、α2、α3、αv、α6、β1、β2、β3的mRNA表達量,western blot法檢測CCN1、p53、pRB和p16蛋白表達量。
3.    Wnt信號通路阻斷劑DKK-1阻斷wnt-3a,DKK-1與wnt-3a共同作用12h后,qRT-PCR法檢測CCN1、p53、p16和MyOD mRNA表達量,western blot法檢測CCN1、p53、pRB和p16蛋白表達量。
4.    尿毒癥小鼠血清刺激:取野生型、假手術和尿毒癥小鼠模型的血清,用DMEM培養液配置成含10%相應組別血清的培養液,分別處理C2C12細胞12h,加等量不含血清的DMEM培養液作為空白對照組。qRT-PCR法檢測CCN1、p53、p16和MyOD mRNA表達量。

結果:
1.    55例終末期腎病患者中符合肌少癥診斷者24例(占43.6%),與無肌少癥組比較顯示低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),低骨骼肌質量指數(t=2.263,P=0.028)為肌少癥可能相關因素。
2.    110例老年人中符合肌少癥診斷者45例(占40.9%),與無肌少癥組組間比較顯示低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),低骨骼肌質量指數(t=9.034,P<0.0001)為肌少癥可能相關因素。
3.    與正常成年人相比,終末期腎病患者血清CCN1(96.35 ± 10.80 ng/ml)、老年人血清CCN1(83.48 ± 8.46 ng/ml)均顯著升高(P<0.05),其中,終末期腎病患者血清CCN1含量較正常成年人升高2.2倍,老年人血清CCN1含量升高1.9倍;與終末期腎病無肌少癥組比較,終末期腎病伴肌少癥患者血清CCN1(119.3 ± 18.17 ng/ml)明顯升高;與老年人無肌少癥組比較,老年人肌少癥患者血清CCN1(104.0 ± 12.17 ng/ml)明顯升高。
4.    在5組不同劑量的小鼠重組wnt-3a分別處理8、12和24h后,與對照組相比,當濃度低于1g/ml時,wnt-3a未引起C2C12細胞活率的顯著變化。而當1.5g/ml 重組wnt-3a處理12和24h后,觀察到C2C12細胞活率明顯下降。
5.    在4組不同劑量的小鼠重組wnt-3a處理12h后,與空白對照組相比,CCN1 mRNA表達量明顯升高,且呈劑量依賴性,隨wnt-3a濃度升高而升高。
6.    1g/ml 重組wnt-3a處理C2C12細胞12h后,與對照組(0g/ml)相比,CCN1、P53和P16 mRNA表達量明顯升高,MyOD mRNA表達量下降,整合素受體α6和β1 mRNA表達量明顯升高。DKK-1抑制劑可明顯抑制wnt-3a上調CCN1表達的作用,并可抑制wnt-3a誘導C2C12細胞衰老的作用,恢復骨骼肌細胞的增殖能力。
7.    在野生型、假手術和尿毒癥小鼠血清條件下生長的C2C12細胞,與野生型血清組相比,尿毒癥血清條件下CCN1,p53和p16 mRNA表達量升高,MyOD mRNA表達量下降。
結論:
1.    老年人肌少癥和終末期腎病肌少癥患者,均伴有低BMI、低肌肉質量分數和低握力,老年人肌少癥還伴有低甘油三酯。
2.    血清CCN1水平與肌少癥相關,老年人肌少癥和終末期腎病肌少癥患者血清CCN1水平較成年人均明顯升高,血清CCN1含量可能是肌少癥的預測指標。
3.    在體外實驗中,一定濃度的wnt-3a可上調C2C12細胞CCN1的表達,誘導細胞衰老過程,同時可抑制細胞增殖能力。
4.    當wnt-3a信號通路被DKK-1抑制后,wnt-3a誘導的CCN1在mRNA和蛋白水平的上調作用被阻斷,被抑制的細胞增殖能力恢復,誘導的衰老指標p53和p16表達量逆轉。
5.    在野生型、假手術和尿毒癥小鼠血清條件下生長的C2C12細胞,尿毒癥血清環境下CCN1表達升高,衰老指標p53和p16升高,細胞增殖指標MyOD下降,表明尿毒癥血清體外仍可誘導C2C12骨骼肌細胞衰老,抑制細胞增殖能力。
關鍵詞:CCN1,肌少癥,wnt-3a,衰老,終末期腎病,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.

目錄
縮略詞表••••••••••••••••••••••••••••••••••••••••••••••••• 1
中文摘要••••••••••••••••••••••••••••••••••••••••••••••••• 3
英文摘要••••••••••••••••••••••••••••••••••••••••••••••••• 6
引言•••••••••••••••••••••••••••••••••••••••••••••••••••••9
材料與方法•••••••••••••••••••••••••••••••••••••••••••••••11
結果•••••••••••••••••••••••••••••••••••••••••••••••••••••25
分析與討論•••••••••••••••••••••••••••••••••••••••••••••••34
參考文獻•••••••••••••••••••••••••••••••••••••••••••••••••37
致謝•••••••••••••••••••••••••••••••••••••••••••••••••••••40
綜述及參考文獻•••••••••••••••••••••••••••••••••••••••••••41
 
縮略詞表
縮略詞    英文全稱    中文全稱
AP    Ammonium persulfate    過硫酸銨
BCA    Bicinchoninic acid    二辛可酸
BMI    Body mass index    體重指數
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    細胞活率測定盒
CKD    Chronic kidney disease    慢性腎臟病
DEPC    Diethypyrocarbonate    焦碳酸二乙酯
DMEM    Dulbecco's Modified Eagle Medium    Dulbecco改良的培養液
DMSO    Dimethyl sulfoxide    二甲基亞砜
DNA    Desoxyribonucleic acid    脫氧核糖核酸
DKK-1    Dickkopf-1    Dickkopf-1
ECM    Extracellular matrix    細胞外基質
EDTA    Ethylene diamine tetraacetic acid    乙二胺四乙酸
ELISA    Enzyme-linked immunosorbent assay    酶聯免疫吸附測定
IGF    Insulin-like growth factor    胰島素樣生長因子
MAPK    Mitogen-activated protein kinases    絲裂原激活蛋白激酶
MMP    Matrix metalloproteinases    基質金屬蛋白酶
PAGE    Polyacrylamide gel electrophoroesis    聚丙烯酰胺凝膠電泳
PBS    Phosphate buffered saline    磷酸鹽緩沖液
PDGF    Platelet-derived growth factor    血小板衍生生長因子
PMSF    Phenylmethanesulfonylfluoride    本甲基磺酰氟
PVDF    Polyvinylidene difluoride    聚偏二氟乙烯
RB    Retinoblastoma protein     視網膜母細胞瘤蛋白
RCC    Renal cell carcinoma    腎細胞癌
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    實時定量聚合酶鏈式反應
Sasp    Senescence-associated secretory phenotype    衰老相關分泌表型
SDS    Sodium dodecyl sulfate    十二烷基磺酸鈉
TBS     Triethanolamine buffered saline    三乙醇胺緩沖鹽水溶液
TEMED    Tetramethylethylenediamine    四甲基乙二胺
TGF-β    Transforming growth factor-β    轉化生長因子-β
Tris    Trishydroxymethylaminomethane    三羥甲基氨基甲烷
VEGF    Vascular endothelial growth factor    血管內皮生長因子
Wnt-3a    Wingless/int1-3a    無翅家族蛋白-3a
YAP    Yes-associated protein    Yes-相關蛋白
 

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