Effect of P62 on the migration and motility of human epidermal cell line HaCaT in high glucose microenvironment and its mechanism
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摘要:
目的 研究高糖微环境下P62蛋白对人表皮细胞株HaCaT迁移和运动性的影响及其可能的分子机制，以探讨糖尿病足创面难愈合的机制。 方法 采用实验研究方法。取对数生长期HaCaT进行实验。取细胞，按随机数字表法（分组方法下同）分为正常对照组（培养液含终物质的量浓度5.5 mmol/L的葡萄糖）及高糖（培养液含终物质的量浓度30.0 mmol/L的葡萄糖）24 h组、高糖48 h组、高糖72 h组。正常对照组细胞行常规培养72 h，高糖72 h组细胞行高糖培养72 h，高糖48 h组细胞先常规培养24 h再高糖培养48 h，高糖24 h组细胞先常规培养48 h再高糖培养24 h后，采用蛋白质印迹法检测P62蛋白表达。取细胞，分为正常对照组、高糖组，分别同前培养48 h后，采用免疫荧光法检测P62蛋白表达（以绿色荧光表示）。取细胞，分为阴性对照小干扰RNA（siRNA）组、P62-siRNA-1组、P62-siRNA-2组、P62-siRNA-3组，并转染相应试剂，于转染后72 h，采用蛋白质印迹法检测P62蛋白表达。取细胞，分为正常糖+阴性对照siRNA组、正常糖+P62-siRNA组、高糖+阴性对照siRNA组、高糖+P62-siRNA组，并行相应处理，于转染后72 h，采用蛋白质印迹法检测P62蛋白表达；行划痕试验检测并计算划痕后24 h细胞迁移率（样本数为9）；在活细胞工作站下，观察3 h内细胞运动范围并计算运动速度（正常糖+阴性对照siRNA组、正常糖+P62-siRNA组、高糖+阴性对照siRNA组、高糖+P62-siRNA组观察细胞数分别为76、75、80、79个）。取细胞，分为正常糖+磷酸盐缓冲液（PBS）组、高糖+PBS组、高糖+N-乙酰半胱氨酸（NAC）组，行相应处理后，于培养48 h，分别采用蛋白质印迹法及免疫荧光法检测P62蛋白表达。除划痕试验外，其余实验各组样本数均为3。对数据行单因素方差分析、LSD检验。 结果 与正常对照组比较，高糖24 h组、高糖48 h组及高糖72 h组细胞P62蛋白表达量均明显增加（P<0.01）。培养48 h，高糖组细胞中P62的绿色荧光强于正常对照组。转染后72 h，与阴性对照siRNA组比较，P62-siRNA-1组、P62-siRNA-2组和P62-siRNA-3组细胞P62蛋白表达量均明显减少（P<0.01）。转染后72 h，与正常糖+阴性对照siRNA组比较，正常糖+P62-siRNA组细胞P62蛋白表达量明显减少（P<0.01），高糖+阴性对照siRNA组细胞P62蛋白表达量明显增加（P<0.01）；与高糖+阴性对照siRNA组比较，高糖+P62-siRNA组细胞P62蛋白表达量明显减少（P<0.01）。划痕后24 h，与正常糖+阴性对照siRNA组［（55±7）%］比较，正常糖+P62-siRNA组细胞迁移率明显升高［（72±14）%，P<0.01］，高糖+阴性对照siRNA组细胞迁移率明显下降［（37±7）%，P<0.01］；与高糖+阴性对照siRNA组比较，高糖+P62-siRNA组细胞迁移率明显升高［（54±10）%，P<0.01］。观察3 h内，高糖+阴性对照siRNA组细胞运动范围较正常糖+阴性对照siRNA组缩小，正常糖+P62-siRNA组细胞运动范围较正常糖+阴性对照siRNA组增大，高糖+P62-siRNA组细胞运动范围较高糖+阴性对照siRNA组增大。与正常糖+阴性对照siRNA组比较，正常糖+P62-siRNA组细胞运动速度明显增加（P<0.01），高糖+阴性对照siRNA组细胞运动速度明显下降（P<0.01）；与高糖+阴性对照siRNA组比较，高糖+P62-siRNA组细胞运动速度明显增加（P<0.01）。培养48 h，与正常糖+PBS组比较，高糖+PBS组细胞P62蛋白表达量明显增加（P<0.01）；与高糖+PBS组比较，高糖+NAC组细胞P62蛋白表达量明显减少（P<0.01）。培养48 h，高糖+PBS组细胞中P62的绿色荧光强于正常糖+PBS组，而高糖+NAC组细胞中P62的绿色荧光弱于高糖+PBS组。 结论 在HaCaT中，高糖微环境可促进P62蛋白表达；敲减P62蛋白可促进其迁移并增加运动性；高糖微环境下活性氧增加可能是P62表达增加的潜在机制。 Abstract:Objective To investigate the effect of P62 on the migration and motility of human epidermal cell line HaCaT in high glucose microenvironment and its possible molecular mechanism, so as to explore the mechanism of refractory diabetic foot wound healing. Methods The method of experimental research was used. HaCaT cells in logarithmic growth phase was taken for experiment. The cells were collected and divided into normal control group (culture solution containing glucose with final molarity of 5.5 mmol/L) and high glucose (culture solution containing glucose with final molarity of 30.0 mmol/L) 24 h group, high glucose 48 h group, and high glucose 72 h group according to the random number table (the same grouping method below). The cells in normal control group were routinely cultured for 72 h, cells in high glucose 72 h group were cultured with high glucose for 72 h, cells in high glucose 48 h group were routinely cultured for 24 h then cultured with high glucose for 48 h, cells in high glucose 24 h group were routinely cultured for 48 h then cultured with high glucose for 24 h. Then the protein expression of P62 was detected by Western blotting. The cells were collected and divided into normal control group and high glucose group. After being correspondingly cultured for 48 h as before, the protein expression of P62 was detected by immunofluorescence method (indicated as green fluorescence). The cells were collected and divided into negative control small interfering RNA (siRNA) group, P62-siRNA-1 group, P62-siRNA-2 group, and P62-siRNA-3 group, and transfected with the corresponding reagents. At post transfection hour (PTH) 72, the protein expression of P62 was detected by Western blotting. The cells were collected and divided into normal glucose+negative control siRNA group, normal glucose+P62-siRNA group, high glucose+negative control siRNA group, and high glucose+P62-siRNA group. After the corresponding treatment, the protein expression of P62 was detected by Western blotting at PTH 72 h, the cell migration rate was detected and calculated at 24 h after scratching by scratch test, with the number of samples being 9; and the range of cell movement was observed and the trajectory velocity was calculated within 3 h under the living cell workstation, with the number of samples being 76, 75, 80, and 79 in normal glucose+negative control siRNA group, normal glucose+P62-siRNA group, high glucose+negative control siRNA group, and high glucose+P62-siRNA group, respectively. The cells were collected and divided into normal glucose+phosphate buffered solution (PBS) group, high glucose+PBS group, and high glucose+N-acetylcysteine (NAC) group. After the corresponding treatment, the protein expression of P62 at 48 h of culture was detected by Western blotting and immunofluorescence method, respectively. Except for scratch test and cell motility experiment, the number of samples was all 3 in the rest experiments. Data were statistically analyzed with one-way analysis of variance and least significant difference test. Results Compared with the protein expression in normal control group, the protein expressions of P62 of cells in high glucose 24 h group, high glucose 48 h group, and high glucose 72 h group were significantly increased (P<0.01). At 48 h of culture, the green fluorescence of P62 of cells in high glucose group was stronger than that in normal control group. At PTH 72, compared with the protein expression in negative control siRNA group, the protein expressions of P62 of cells in P62-siRNA-1 group, P62-siRNA-2 group, and P62-siRNA-3 group were significantly decreased (P<0.01). At PTH 72, compared with the protein expression in normal glucose+negative control siRNA group, the protein expression of P62 of cells in normal glucose+P62-siRNA group was significantly decreased (P<0.01), while the protein expression of P62 of cells in high glucose+negative control siRNA group was significantly increased (P<0.01); compared with the protein expression in high glucose+negative control siRNA group, the protein expression of P62 of cells in high glucose+P62-siRNA group was significantly decreased (P<0.01). At 24 h after scratching, compared with (55±7)% in normal glucose+negative control siRNA group, the cell migration rate in normal glucose+P62-siRNA group was significantly increased ((72±14)%, P<0.01), while the cell migration rate in high glucose+negative control siRNA group was significantly decreased ((37±7)%, P<0.01); compared with that in high glucose+negative control siRNA group, the cell migration rate in high glucose+P62-siRNA group was significantly increased ((54±10)%, P<0.01). Within 3 h of observation, the cell movement range in high glucose+negative control siRNA group was smaller than that in normal glucose+negative control siRNA group, while the cell movement range in normal glucose+P62-siRNA group was larger than that in normal glucose+negative control siRNA group, and the cell movement range in high glucose+P62-siRNA group was larger than that in high glucose+negative control siRNA group. Compared with that in normal glucose+negative control siRNA group, the cell trajectory speed in normal glucose+P62-siRNA group was significantly increased (P<0.01), while the cell trajectory speed in high glucose+negative control siRNA group was significantly decreased (P<0.01); compared with that in high glucose+negative control siRNA group, the cell trajectory speed in high glucose+P62-siRNA group was significantly increased (P<0.01). At 48 h of culture, compared with that in normal glucose+PBS group, the protein expression of P62 of cells in high glucose+PBS group was significantly increased (P<0.01); compared with that in high glucose+PBS group, the protein expression of P62 of cells in high glucose+NAC group was significantly decreased (P<0.01). At 48 h of culture, the green fluorescence of P62 of cells in high glucose+PBS group was stronger than that in normal glucose+PBS group, while the green fluorescence of P62 of cells in high glucose+NAC group was weaker than that in high glucose+PBS group. Conclusions In HaCaT cells, high glucose microenvironment can promote the protein expression of P62; knockdown of P62 protein can promote the migration and increase the mobility of HaCaT cells; and the increase of reactive oxygen species in high glucose microenvironment may be the underlying mechanism for the increase of P62 expression. -
Key words:
- Diabetic foot /
- Reactive oxygen species /
- Cell migration assays /
- P62 /
- High glucose /
- Wound repair
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证明超声清创法可显著减轻烧伤残余创面的细菌负荷，减少术后血肿形成，促进移植皮片成活，从而缩短患者病程。
烧伤，尤其是深度烧伤会遗留严重瘢痕，对患者身心造成损害［1, 2, 3］。深度烧伤创面难以愈合或一次封闭困难，均会导致残余创面形成。临床上修复残余创面常采用清创+植皮术，而传统清创手术易误伤创面周围健康组织［4］，违背现代精准医学的治疗理念。近年来，超声清创技术在糖尿病足、下肢静脉溃疡、压疮、骨外露等创面修复［5, 6, 7, 8］中取得了良好效果，但目前国内鲜见将超声清创技术应用于烧伤残余创面的相关报告。解放军联勤保障部队第980医院（以下简称本单位）近年来将超声清创技术应用于烧伤残余创面，取得了良好的临床效果。
1. 对象与方法
本回顾性队列研究符合《赫尔辛基宣言》的基本原则，根据本单位伦理委员会政策，临床资料可以在不泄露患者身份信息的前提下分析、使用。
1.1 入选标准
纳入标准：（1）原发深Ⅱ度或Ⅲ度烧伤经清创植皮后仍存在残余创面；（2）创面分布相对集中，创面总面积≥1%TBSA，且至少存在1处残余创面直径≥5 cm。（3）对残余创面行传统清创法或超声清创法+植皮治疗者。排除标准：资料不全者。
1.2 患者一般资料及烧伤创面情况
2017年8月—2021年8月，本单位收治64例符合入选标准的患者，其中男41例、女23例，年龄为9个月~67岁，平均年龄31岁，烧伤总面积25%~90%TBSA，残余创面总面积1%~17%TBSA，致伤原因：火焰烧伤者36例，热液烫伤者23例，电烧伤者5例。根据创面清创方法，将患者分为超声清创组（34例）和传统清创组（30例）。2组患者性别、年龄、致伤原因、烧伤部位等一般情况比较，差异均无统计学意义（P>0.05）；2组患者烧伤总面积、残余创面面积、伤后清创时间等创面情况均相似（P>0.05）。见表1。清创前，2组患者的所有创面中均有细菌感染：超声清创组创面中检出金黄色葡萄球菌者12例、铜绿假单胞菌者15例、鲍曼不动杆菌者5例、粪肠球菌者3例、阴沟肠杆菌者2例，其中复合感染者3例；传统清创组创面检出金黄色葡萄球菌者11例、铜绿假单胞菌者15例、鲍曼不动杆菌者3例、肺炎克雷伯菌者2例、阴沟肠杆菌者2例，其中复合感染者3例。
表1 2组烧伤残余创面患者一般资料及其烧伤创面情况比较组别 例数 性别（例） 年龄（岁， 致伤原因（例） 烧伤部位（例） 烧伤总面积（%TBSA， 残余创面面积（%TBSA， 伤后清创时间（d， 男 女 火焰烧伤 热液烫伤 电烧伤 头面颈部 前后躯干 四肢 超声清创组 34 22 12 31 19 12 3 4 20 10 51 10.9 32 传统清创组 30 19 11 32 17 11 2 3 19 8 50 10.1 33 统计量值 χ2=0.01 t=0.30 χ2=0.10 χ2=0.14 t=0.29 t=0.89 t=0.71 P值 0.909 0.972 0.949 0.932 0.707 0.907 0.169 注：TBSA为体表总面积 1.3 治疗方法
2组患者入院后均行常规补液、换药等治疗，术前纠正电解质紊乱、低蛋白血症、贫血、血糖和血压异常等情况，选择敏感抗生素防治感染，改善创面微循环。术中全身麻醉下对创面进行清创：（1）超声清创组创面，采用超声清创机根据创面大小使用500~2 000 mL生理盐水清除坏死组织或陈旧性肉芽至新鲜点状出血，清创后以1∶250 000肾上腺素盐水浸润的纱布覆盖创面以减少出血。超声机功率设置（50%~80%额定功率）及清创时长根据肉芽生长、坏死组织残存及感染情况确定。（2）传统清创组创面，采用传统锐性清创的方式，清创同时以1∶250 000肾上腺素盐水浸润的纱布湿敷，必要时行电凝止血。清创结束后，2组创面均使用过氧化氢、氯已定、生理盐水等冲洗3~5遍后植皮。功能部位或对容貌影响较大的暴露区创面首选大张皮片植皮；非功能部位创面，尤其是供皮困难者，选择移植邮票皮。植皮后四肢等部位采用无菌纱布适当加压包扎；面部等不易包扎的部位予缝线打包加压固定。术后行抗生素治疗，每2~3天换药1次。对于Ⅰ期手术后未愈合创面行二次清创+植皮术，2组创面的清创方式均分别同其Ⅰ期。
1.4 观察指标
1.4.1 创面分泌物细菌阳性率
术后3 d，于创面分泌物较多处选取标本行微生物培养及药物敏感试验检测细菌情况并计算细菌阳性率。创面细菌阳性率=细菌阳性患者例数÷患者总例数×100%。
1.4.2 皮片成活率及皮下血肿发生率
术后7 d，对于采用邮票皮治疗者，若面积较大，则统计5处约5 cm×5 cm植皮区内成活皮片数量；若面积较小，则统计所有植皮区域内成活皮片数量［4］。对于采用大张皮植皮者，行大体观察直接评估皮片成活情况。皮片成活标准为贴附良好、颜色红润、按压后充血明显、无明显滑动。皮片成活率=成活皮片数÷移植皮片总数×100%。术后7 d，大体观察创面皮下血肿发生率，皮下血肿发生率=皮下血肿形成患者例数÷患者总例数×100%。
1.4.3 创面愈合时间及二次清创率
出院时，统计患者创面愈合时长（二次清创者以Ⅱ期创面愈合时间为准）及清创次数并计算二次清创率，二次清创率=二次清创患者例数÷患者总例数×100%。
1.5 统计学处理
采用SPSS 17.0统计软件对数据进行分析。符合正态分布的计量资料数据以
2. 结果
2.1 创面分泌物细菌阳性率
术后3 d，超声清创组创面感染金黄色葡萄球菌者2例、铜绿假单胞菌者2例，传统清创组创面感染金黄色葡萄球菌者5例、铜绿假单胞菌者3例、鲍曼不动杆菌者1例、肺炎克雷伯菌者1例、阴沟肠杆菌者1例；超声清创组患者创面细菌阳性率为11.8%（4/34）明显低于传统清创组的36.7%（11/30），χ2=5.51，P=0.019。
2.2 皮片成活率及皮下血肿发生率
术后7 d，超声清创组创面的移植皮片成活率为（92
2.3 创面愈合时间及二次清创率
出院时，超声清创组创面愈合时长为（11.0
2.4 典型病例
患者男，38岁，致伤原因为电烧伤，全身多处均为Ⅲ度烧伤，总面积为26%TBSA，主要位于左下肢（14%TBSA）及头面部（3%TBSA）、左上肢（3%TBSA）、背部（4%TBSA）、左臀部（2%TBSA），伤后被送至当地医院治疗，其间行全身多处创面削痂+Meek植皮术。患者于伤后25 d转入本单位。入院时，患者部分创面已经基本封闭，已愈合区域与肉芽创面相互交错；残余创面主要位于背部，面积为2%TBSA，其表面可见肉芽组织增生及稀薄分泌物。入院后，从患者创面分泌物中检出金黄色葡萄球菌，后续采用静脉输注抗生素抗感染、纠正电解质紊乱等措施改善患者全身状况，创面每2~3天换药1次。入院后第7天，在全身麻醉下采用超声清创机对患者背部残余创面进行清创，清创机功率设置为75%额定功率，使用约500 mL生理盐水冲洗创面。然后选择右侧背部皮肤为供皮区，切取相应大小刃厚皮片行邮票皮移植术并采用无菌纱布适当加压包扎。术后行抗生素治疗，每2~3天换药1次。术后3 d，取创面中少量分泌物行微生物培养，未检出细菌。术后7 d，创面皮片成活率约为98%，皮片下无明显血肿。术后12 d创面完全愈合。见图1。
1 超声清创法+邮票皮移植治疗背部烧伤患者残余创面的疗效。1A.伤后25 d入院时，创面中较少肉芽组织增生；1B.伤后32 d背部创面清创术前，见肉芽组织增生明显，突出皮面；1C.清创术中；1D.术后12 d，邮票皮片存活良好3. 讨论
浅Ⅱ度烧伤经适当处理后可愈合，但深Ⅱ度及Ⅲ度等深度烧伤创面愈合困难，勉强愈合后也会遗留瘢痕或反复破溃，严重影响机体外观和功能［9, 10, 11］。因此，对深度烧伤常在早期行外科手术修复，常形成残余创面需在Ⅱ期行清创植皮术。理想的清创方式应具备高效且尽量保留健康组织、减少出血、降低住院费用、减轻创面中细菌负荷及提高愈合率等特点［12］。烧伤残余创面存在已愈合区域与坏死组织或肉芽组织相互交叉、镶嵌分布、边缘不规则等特点，清创难度大，传统锐性清创法的应变效能差，会造成周围正常组织不必要的损伤，出血较多。这导致经验较少的年轻医师在治疗残余创面的过程中存在较多突出问题［4］。
超声清创法是近年来兴起的清创方式，为残余创面清创提供了新的选择［13］。高频超声以热效应为主，低频超声以机械效应为主，即分别具有空化效应与声流作用［14, 15, 16］。超声清创机采用低频、高能超声波加载喷射流技术，在冲洗创面过程中会产生空化效应，即通过超声波形成的空化泡崩塌产生的微射流及高压可清除坏死组织［17, 18, 19］。超声清创机操作灵活，可用于平面或腔隙等不同类型创面；还可根据坏死组织或肉芽组织情况选择不同的功率，达到最佳清创效果。同时，超声清创机可将液体以雾化的形式喷出，使液体全面接触创面以更精确地清除非活性组织，又避免了对周围正常组织的附带损伤［5，17，20］。
创面细菌负荷的减轻程度是反映清创效果的一个重要指标。创面细菌负荷与细菌生物膜形成密切相关，生物膜可保护细菌免受抗生素、防腐剂和宿主免疫的影响，还可逐步释放浮游细菌导致创面持续感染，严重影响创面皮片成活率［21, 22, 23, 24, 25, 26］。有研究显示超声不仅对革兰阳性、革兰阴性、需氧和厌氧细菌具有明显抗菌作用［27, 28］，还可以有效清除细菌生物膜［21］，显著减少细菌负荷，改善创面条件［29, 30, 31］。本研究结果显示，超声清创组术后3 d创面细菌阳性率显著低于传统清创组，同时前者的二次清创率也显著低于后者，提示超声清创法在清除创面坏死组织，减轻细菌负荷等方面具有显著作用，其结果与前述既往国内外研究结果一致。
此外，超声清创法可有效减轻术后炎症反应和疼痛，还可促进伤口组织中碱性FGF和EGF的高表达，改善创面微循环，促进血管生成和胶原沉积，提升组织再生和修复能力，这可能与超声产生的空化效应和声流作用等机械能刺激信号转导，继发广泛的细胞效应有关［18, 19，32, 33, 34］。本研究中超声清创组的皮片成活率显著高于传统清创组，且创面愈合时间显著短于传统清创组。移植皮片的成活率与皮下积血、积液等密切相关。相关研究指出，超声清创机可通过热效应达到减少出血的效果［5，35, 36］，但由于术中出血与组织液、膨胀液或冲洗液等混合在一起，难以精确估计出血量。但是本研究显示，超声清创组皮下血肿发生率显著低于传统清创组，提示减少术中出血和皮下血肿形成可能是超声清创法提高皮片成活率，促进创面愈合的机制之一。
综上，与传统清创法相比，超声清创法可在烧伤残余创面清创中彻底清除坏死组织和陈旧性肉芽组织、减轻细菌负荷、降低皮下血肿发生率，促进移植皮片成活，从而缩短创面愈合时间，为烧伤残余创面的修复提供了新的方法和选择。超声清创法对于提高烧伤残余创面的修复效果具有重要意义，值得临床推广。
张喻平：实验操作、论文撰写；张琼、邓芳：数据整理、统计学分析；陈兵、张均辉、胡炯宇：研究指导、论文修改、经费支持所有作者均声明不存在利益冲突 -
参考文献
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1 蛋白质印迹法检测正常对照组和高糖培养3个时间点组HaCaT中P62蛋白表达
注：GAPDH为3-磷酸甘油醛脱氢酶；条带上方1、2、3、4分别指示正常对照组、高糖24 h组、高糖48 h组、高糖72 h组
2 正常对照组和高糖组HaCaT培养48 h的P62蛋白表达 异硫氰酸荧光素-4'，6-二脒基-2-苯基吲哚×630。2A、2B、2C.分别为正常对照组P62染色、细胞核染色、P62与细胞核染色重叠图片，细胞核完整，P62蛋白表达较少，主要分布于细胞质；2D、2E、2F.分别为高糖组P62染色、细胞核染色、P62与细胞核染色重叠图片，细胞核完整，P62蛋白表达较图2A多
注：P62阳性染色为绿色，细胞核阳性染色为蓝色
3 蛋白质印迹法检测转染siRNA后4组HaCaT中P62蛋白表达
注：GAPDH为3-磷酸甘油醛脱氢酶，siRNA为小干扰RNA；条带上方1、2、3、4分别指示阴性对照siRNA组、P62-siRNA-1组、P62-siRNA-2组、P62-siRNA-3组
4 蛋白质印迹法检测高糖微环境下siRNA对4组HaCaT转染后72 h的P62蛋白表达的作用
注：GAPDH为3-磷酸甘油醛脱氢酶，siRNA为小干扰RNA；条带上方1、2、3、4分别指示正常糖+阴性对照siRNA组、正常糖+P62-siRNA组、高糖+阴性对照siRNA组、高糖+P62-siRNA组
5 4组HaCaT处理完成后划痕后各时间点细胞划痕面积情况 倒置光学显微镜×100。5A、5B、5C、5D.分别为正常糖+阴性对照小干扰RNA（siRNA）组、正常糖+P62-siRNA组、高糖+阴性对照siRNA组、高糖+P62-siRNA组划痕后0 h（即刻），划痕情况相近；5E、5F、5G、5H.分别为正常糖+阴性对照siRNA组、正常糖+P62-siRNA组、高糖+阴性对照siRNA组、高糖+P62-siRNA组划痕后24 h，图5F划痕面积明显小于图5E，图5G划痕面积明显大于图5E，图5H划痕面积明显小于图5G
6 活细胞工作站观察4组HaCaT 3 h内的运动范围。6A、6B、6C、6D.分别为正常糖+阴性对照siRNA组、正常糖+P62-siRNA组、高糖+阴性对照siRNA组、高糖+P62-siRNA组，其中图6B、6D运动范围分别较图6A、6C明显增大
注：siRNA为小干扰RNA；细胞运动起点均为坐标（0，0），运动终点为位于4个象限中的圆点，连接两者之间的曲线为细胞运动轨迹
7 蛋白质印迹法检测高糖微环境下NAC对3组HaCaT培养48 h的P62蛋白表达的影响
注：GAPDH为3-磷酸甘油醛脱氢酶，PBS为磷酸缓冲盐溶液，NAC为N-乙酰半胱氨酸；条带上方1、2、3分别指示正常糖+PBS组、高糖+PBS组、高糖+NAC组
8 3组HaCaT培养48 h的P62蛋白表达 异硫氰酸荧光素-4'，6-二脒基-2-苯基吲哚×630。8A、8B、8C.分别为正常糖+PBS组P62染色、细胞核染色、P62与细胞核染色重叠图片，细胞核完整，P62蛋白表达较少，主要分布于细胞质；8D、8E、8F.分别为高糖+PBS组P62染色、细胞核染色、P62与细胞核染色重叠图片，细胞核完整，P62蛋白表达较图8A多；8G、8H、8I.分别为高糖+NAC组P62染色、细胞核染色、P62与细胞核染色重叠图片，细胞核完整，P62蛋白表达较图8D少
注：P62阳性染色为绿色，细胞核阳性染色为蓝色；PBS为磷酸缓冲盐溶液，NAC为N-乙酰半胱氨酸
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