| A型肉毒毒素影响透明质酸钠凝胶代谢的实验研究 | |
| Alternative Title | Experimental Study on the Effect of Botulinum Toxin Type A on the Metabolism of Hyaluronic Acid Gel |
| 张珍珍 | |
| Subtype | 硕士 |
| Thesis Advisor | 张选奋 |
| 2020-05-28 | |
| Degree Grantor | 兰州大学 |
| Place of Conferral | 兰州 |
| Degree Name | 医学硕士 |
| Degree Discipline | 外科学 |
| Keyword | 透明质酸钠凝胶 A型肉毒毒素 透明质酸酶 联合使用 面部年轻化 |
| Abstract | 背景：A型肉毒毒素（Botulinum toxin type A,BTX-A）和透明质酸钠（Hyaluronic acid,HA）凝胶联合注射治疗面部年轻化的疗效较单独注射HA凝胶更好，同时发现，BTX-A能明显降低体内HA凝胶的代谢率，具有显著协同作用，但目前国内外并无相关文献报道BTX-A降低HA凝胶代谢的相关机制，另外，在临床操作中，因HA填充剂量过大、填充物移位、血管栓塞、皮肤组织坏死、失明等并发症的发生率较高，需及时注射透明质酸酶（Hyaluronidase,HAase）进行溶解，但目前尚无BTX-A对HAase活性影响的相关文献报道。 目的：本实验拟初步研究BTX-A对体外HA凝胶酶降解反应的影响，并在大鼠体内观察BTX-A对HA凝胶代谢的影响，进而探讨二者联合治疗协同作用的发生机制，为临床操作提供实验依据。 方法：1、BTX-A对体外HA凝胶酶解反应的影响取海薇和润百颜HA凝胶各5份，每份0.05mL，经2%甲苯胺蓝染色12h，室温（25℃）复温1h后，分别加入BTX-A 2.5U（2.5U/mL）、5.0U（5.0U/mL）、10.0U（10.0U/mL）、20.0U（20.0U/mL）及生理盐水1.0mL，再加入300.0UHAase，于显微镜下观察HA凝胶的酶解状态，记录达最大酶解状态的时间，在0-110min，每间隔10min由显微镜成像系统采集图片，经ImageJ软件计算平均光密度值（OD）。 2、BTX-A对大鼠体内HA凝胶代谢的影响在大鼠背部两侧皮下各注射润百颜HA凝胶0.25mL，5分钟后于BTX-A组真皮层注射BTX-A 5.0U（5.0U/0.1mL），右侧注射生理盐水0.1mL作为自身对照。分别在3d、7d、14d、30d、60d、90d对注射区域皮肤组织及剩余HA凝胶进行取材。免疫组化法检测皮肤组织中透明质酸酶-1（HYAL-1）和透明质酸酶-2（HYAL-2）的表达，采用黄嘌呤氧化酶法和紫外比色法分别检测组织匀浆中SOD和CAT的活性，采用分光光度法检测MDA的含量，利用排水法测量剩余HA凝胶体积，并计算代谢率。 结果：1、BTX-A对体外HA凝胶酶解反应的影响 在0U、2.5U、5.0U、10.0U、20.0U BTX-A作用下，润百颜产品分别在60min、70min、80min、100min、100min达到最大酶解状态，酶解率分别为72.149%、68.791%、64.683%、58.599%、58.334%海薇产品分别在60min、70min、90min、100min、100min达到最大酶解状态，酶解率分别为63.390%、54.999%、44.965%、41.890%、42.067%。在0-10.0U，随着BTX-A剂量的增加，对HAase活性的抑制作用逐渐增强，两种HA凝胶达最大酶解状态的时间延长，酶解速率逐渐减小，呈剂量依赖性超过10.0U，抑制作用不再增加。 2、BTX-A对大鼠体内HA凝胶降解的影响 一般情况各动物在实验过程中日常活动、生活习惯无明显改变，无躁动及倦怠等异常表现，进食水正常实验注射前各组大鼠背部皮肤完整，两侧对称无隆起，注射药物后两侧注射区域局部隆起，触之软，界限清，活动度较小，无红斑、皮肤破溃等改变BTX-A组隆起第3d、7d、14d与对照组相同，第30d、60d、90dBTX-A组较对照组明显隆起。 皮肤组织中HYAL-1、HYAL-2的表达 BTX-A注射后第3d，皮肤组织中HYAL-1的OD值从对照组的0.256±0.005下降到0.243±0.003（t=3.911，P=0.008）第7d OD值从对照组的0.254±0.006下降到0.240±0.006（t=2.846，P=0.029）14d及以后，两组组织中HYAL-1的表达无变化（P>0.05）。BTX-A注射后第3d，皮肤组织中HYAL-2的OD值从对照组的0.264±0.010下降到0.238±0.005（t=3.897，P=0.008）第7d OD值从对照组的0.263±0.003下降到0.236±0.003（t=11.179，P<0.001）第14d OD值从对照组的0.258±0.006下降到0.233±0.003（t=6.165，P=0.001）30d及以后，两组组织中HYAL-2的表达无变化（P>0.05）。 组织中SOD、CAT活性及MDA含量测定 BTX-A注射后皮肤组织中SOD、CAT的活性及MDA的含量均无变化（P>0.05）。 剩余HA凝胶体积及代谢率 BTX-A组和对照组HA凝胶初始体积分别为447.5±10.9mm3和440.0±14.1mm3（t=0.728,P=0.496），90d后分别下降到305.0±11.2mm3和197.5±10.9mm3（t=11.926，P<0.001），代谢率分别为31.8%和55.1%。BTX-A组代谢在第7d后较对照组即显著减慢（P<0.001）。 结论： 1、BTX-A在0-10.0U对HA的酶解具有剂量依赖性抑制作用。 2、BTX-A降低大鼠体内HYAL-1（3d、7d）和HYAL-2（3d、7d、14d）的表达，显著降低体内HA凝胶的代谢。 3、BTX-A联合HA凝胶治疗，先行注射BTX-A，14d后再行注射HA凝胶。当选择同期注射BTX-A，在HA填充注射后14d内需注射HAase溶解时，需酌情加大HAase的剂量。 关键词：透明质酸钠凝胶，A型肉毒毒素，透明质酸酶，联合使用，面部年轻化 |
| Other Abstract | Background Botulinum toxin type A(BTX-A) and Hyaluronic acid(HA) gel combined with injection for treating facial rejuvenation is better than HA gel injection alone.At the same time, it was found that BTX-A can significantly reduce the metabolic rate of HA gel in the body and has a significant synergistic effect. However, there is no related literature at home and abroad to report the related mechanism of BTX-A reducing HA gel metabolism. In addition, in clinical operations, the incidence of HA injection complications such as excessive fillerdose, fillerdisplacement, vascular embolism, skin tissue necrosis, blindness, etc. is higher. And it is necessary to inject hyaluronidase(HAase)in time to dissolve, but there is no related literature about the effect of BTX-A on the activityof HAase. Objectives This experiment intends to study the effect of BTX-A on the degradation of HA gel in vitro and observe the effect of BTX-A on the metabolism of HA gel in rats, and to explore the mechanism of the synergistic effect of the two combined treatments, and to provide experimental basis for clinical operations. Methods Effect of BTX-A on the enzymatic hydrolysis of HA gel in vitro Take 5 parts of Matrifill and Biohyalux HA gel each with a volume of 0.05mL, after staining with 2% toluidine blue for 12 hours and re-warming at room temperature ,25℃,for 1 hour, add BTX-A 2.5U (2.5 U/ mL), 5.0U (5.0U / mL), 10.0U (10.0U / mL), 20.0U (20.0U / mL), and normal saline 1.0mL, respectively. Then add 300.0U HAase, and immediately observe the enzymolysis status of the HA gel under a microscope, and record the time to reach the maximum enzymatic hydrolysis state,and collect Images by the microscope imaging system at intervals of 0 to 110 min, and calculatethe average optical density (OD)valuesby ImageJ software. Effect of BTX-A on the degradation of HA gel in rats The HA filler, Biohyalux,was injected subcutaneously on both sides of the back of the rats with a volume of 0.25 mL.after 5 minutes, BTX-A was injected into the dermis layer of the left at a dose of 5.0U (5.0U/0.1mL) as the BTX-Agroup, and 0.1mL of normal saline was injected on the right as the control group.On the 3rd, 7th, 14th, 30th, 60th, and 90th days, the skin tissues and the remaining HA gel were collected from the injection area for detecting the expression of HYAL-1 and HYAL-2 in the skin tissues by immunohistochemical staining, detecting the activity of superoxide dismutase(SOD) by xanthine oxidase method, detecting the activity of catalase(CAT) by ultraviolet colorimetry, and detecting the content of malondialdehyde(MDA)by spectrophotometry, and calculating the remaining volume of HA gel by the drainage method, and calculating the metabolic rate of HA gel. Results 1. Effect of BTX-A on the enzymatic hydrolysis of HA gel in vitro Under the action of 0U, 2.5U, 5.0U, 10.0U, 20.0U BTX-A, TheHA fillerof Biohyaluxreached the maximum enzymatic hydrolysis state at 60min, 70min, 80min, 100min, and 100min, and the enzymatic hydrolysis rates were 72.663%, 68.791%, 64.683%, 58.599%, 58.334%,respectively.TheHA fillerof Matrifillreached the maximum enzymatic hydrolysis state at 60min, 70min, 90min, 100min, and 100min, and the enzymolysis rates were 63.390%, 54.999%, 44.965%, 41.890%, 42.067%,respectively.The dosage of BTX-A is 0-10.0U,with the increase of the dosage, the inhibitory effect on the activity of HAase is gradually increased. The time for the two HA gels to reach the maximum enzymatic hydrolysis state is prolonged, and the enzymatic hydrolysis rate gradually decreases, showing a dose-dependent.When the dose of BTX-A exceeds 10.0U, the inhibitory effect no longer increases. 2. Effect of of BTX-A on the degradation of HA gel in rats During the experiment, there was no obvious change in daily activities and living habits of each animal, no abnormal performance such as agitation and burnout, and normal eating and drinking. Before the injection, the skin of the back of each group of rats was intact, and the sides were symmetrical without bulge.After injection of the drugs, the injection area is locally bulged, which has the characteristics of soft touch, clear boundaries, and low mobility, without changes such as erythema and skin ulceration.On the 3rd, 7th, and 14th days of the experiment, there was no significant difference in the bulge between the test group and the control group, and the bulge was significantly higher on the 30th, 60th, and 90th day than in the control group. On the 3rd day after BTX-A injection, the OD value of HYAL-1 in the skin tissue decreased from 0.256 ±0.005 to 0.243 ±0.003 in the control group (t= 3.911, P= 0.008).on the 7th day, the OD value in the BTX-Agroupdecreased from 0.254 ±0.006 to 0.240 ±0.006in the control group. (t= 2.846, P= 0.029).after 14 days and later, there was no change in the expressionof HYAL-1 in the two groups (P>0.05). On the 3rd day after BTX-A injection, the OD value of HYAL-2 in theskin tissue decreased from 0.264 ±0.010 to 0.238 ±0.005 in the control group (t= 3.897, P= 0.008).on the 7th day, the OD value in the BTX-Agroupdecreased from 0.263 ±0.003 to 0.236 ±0.003 in the control group.(t= 11.179, P<0.001). On the 14th day, the OD value in the BTX-Agroupdecreased from 0.258 ±0.006 to 0.233 ±0.003 in the control group (t= 6.165, P= 0.001). After 30 days, there was no change in the expression of HYAL-2 in the two groups. (P>0.05). There was no significant difference in the activity of SODandCATbetween the BTX-A groupand the control group (P>0.05),and there was no significant difference inthe content of MDA between the BTX-A groupand the control group (P>0.05). The initial volume of HA gel in the BTX-A group and the control group was 447.5 ±10.9mm3and 440.0 ±14.1mm3(t= 0.728, P= 0.496). After 90 days, the remaining volume was reduced to 305.0 ±11.2mm3and 197.5 ±10.9mm3(t= 11.926, P<0.001), and the metabolic rates were 31.8% and 55.1%, respectively. The metabolic rate of the two groups showed a significant difference after 7 days (P<0.001). Conclusions BTX-A has a dose-dependent inhibitory effect on the enzymatic hydrolysisof HA in the dose range of 0-10.0U. BTX-A can reduce the expression of HYAL-1（3d, 7d）and HYAL-2（3d, 7d, 14d）in rats and significantly reduce the metabolism of HA gel in vivo. Combined injection of BTX-A and HA gel to treat facial rejuvenation, BTX-A was injected first and HA gel was injected 14 days later.When BTX-A is chosen to be injected at the same time and the HAase needs to be dissolved within 14 days after the HA fillerinjection, the dose of HAasemay need to be increased. Key words: Hyaluronic acid gel, Botulinum toxin type A, Hyaluronidase, Combined injection, Facial rejuvenation |
| Pages | 65 |
| URL | 查看原文 |
| Language | 中文 |
| Document Type | 学位论文 |
| Identifier | https://ir.lzu.edu.cn/handle/262010/463943 |
| Collection | 第二临床医学院 |
| Affiliation | 第二临床医学院 |
| Recommended Citation GB/T 7714 | 张珍珍. A型肉毒毒素影响透明质酸钠凝胶代谢的实验研究[D]. 兰州. 兰州大学,2020. |
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