清热、活血、益气法对创伤组织修复的临床与实验研究
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摘要
围手术期是外科非常重视的热点问题之一,日益得到重视。创伤修复的目标从追求愈合速度逐渐转变为重视愈合质量,组织修复的内涵正逐步扩大,研究的范围由体表深入到体内,由局部到全身,由宏观到微观。现代医学认为,创伤修复不仅有形态学的变化,也有复杂的生化学变化,生长因子和胶原在修复过程中起到重要作用。目前研究较多的有成纤维细胞生长因子(FGF),Ⅰ,Ⅲ型胶原。
中医认为:创伤打击致使机体气血凝滞、营气不从,经络阻塞,脏腑失和;导致郁而化热、瘀血停滞、正气亏虚等。机体修复过程有明显的时段性,早期患者多表现为热证和血瘀证,中期患者多表现为虚证和血瘀证,术后康复期多表现为虚证。中药可以多靶点、多环节地发挥作用。近年有关创伤修复的逐步展开,但对中医药在创伤修复过程中的作用规律及机理仍缺乏深入研究。
本研究试图在中医理论指导下,整体思考创伤修复的变化规律,根据既往的临床实践,通过临床研究,分析活血、益气、清热法对创伤组织修复不同阶段的的作用。结合临床资料总结,探讨围手术期不同时段的中医辩证用药的共性及其优势。并通过创伤修复实验研究,检测碱性成纤维细胞生长因子(basic fibroblast growth factor,bFGF)mRNA表达的关系、观察各组大鼠创面修复早期肉芽组织中Ⅰ、Ⅲ型胶原的变化;来探讨清热、活血、益气法改善大鼠伤口愈合的作用,分析其促进创面愈合的实际作用与机理。
临床研究
选择开放胆囊切除术的病例,随机分组。两组病人在性别、年龄、手术时间、麻醉时间、出血量、胆汁细菌培养等方面比较,P均＞0.05,差异无统计学意义,具有可比性。对照组按照常规临床路径治疗;治疗组方案在标准临床路径的基础上加用分期中医辩证处方治疗方案:手术当日及术后第1-3天静滴清开灵20ml+生理盐水100ml,丹参针20ml+生理盐水100ml;术后第4-7天静滴参麦针20ml+生理盐水100ml,丹参针20ml+生理盐水100ml。观察患者术后体温恢复正常时间、伤口愈合情况、白细胞恢复正常时间,胃肠功能恢复时间、体重变化、伤口愈合情况、胃肠道功能回复情况、并发症情况、住院时间、住院费用等指标。
临床研究结果:治疗组在体温恢复正常时间、血WBC恢复正常时间、肛门排气时间、术后首次排便时间、拆线时间、体重减轻等方面,均优于对照组(P＜0.05);治疗组并发症(肺部感染、泌尿系感染、伤口感染裂开)发生率低于对照组;治疗组并不增加患者住院费用。
实验研究
实验动物及分组SPF级SD大鼠,雄性,随机分组,创面造模后喂药分第3天、第7天两个观测时段。每个时段又分为模型组(生理盐水)、清热+活血组、益气+活血组。根据成人日服用药量的5倍计算大鼠腹腔注射剂量,用药后第3天、7天(每组8-13号动物),取新生创面肉芽组织,送检。
基本观察:术后丹参+清开灵组在造模后1—3天进食量、体重减轻优于模型组;丹参+参麦灵组在造模后4—7天进食量、体重增加优于模型组(P＜0.05)。
实验结果(以下P＜0.05或P＜0.01)):
清开灵+丹参组用药3天后,肉芽生长明显红活,创面缩小程度显著;HE染色表现:镜下见创伤表面血痂形成,痂下可见渗出的液体及炎症细胞(图5),各例组织均可见肉芽组织修复性增生的改变(图6)。免疫组化:镜下见各例组织中均有少量至中等量黄褐色Ⅰ型胶原阳性表达。镜下见各例组织中均有少量黄褐色Ⅲ型胶原弱阳性表达;bFGF mRNA表达比造模组增加显著。七天后表现为:进食、活动等逐渐恢复,丹参+清开灵组肉芽组织明显向心性、团块状、瘤性增生,创面由于为来自不同方向生长肉芽组织而体积变大,将创面完全封闭,但组织不平整;HE染色表现:镜下仍可见创伤表面血痂形成,痂下少量渗出的液体及炎症细胞(图7),肉芽组织修复性增生明显(图8)。免疫组化:镜下所见与3天时相似,少至中等量Ⅰ型胶原阳性表达。镜下所见与3天时相似,少量Ⅲ型胶原弱阳性表达,部分组织内有少量黄褐色Ⅲ型胶原弱阳性表达。相对于用药3天,清开灵+丹参组在7天时bFGF mRNA基因表达明显增加。
参麦+丹参组用药3天后,肉芽生长较均匀,创面缩小;HE染色表现:镜下见创面肉芽组织增生伴较多炎细胞浸润(图9)。免疫组化:镜下见各例组织中均有黄褐色Ⅰ型胶原弱阳性表达。少量黄褐色Ⅲ型胶原弱阳性表达。bFGF mRNA表达明显增加。七天后,增生出的肉芽组织明显平整,部分增生组织平整创口处被修复;HE染色表现:镜下所见与3天相似,镜下可见较多肉芽组织增生;免疫组化:镜下见各例组织中Ⅰ型胶原表达较3天时明显,个别组织出现Ⅰ型胶原强阳性表达。镜下见各例组织中Ⅲ型胶原表达较3天时明显增多,但未见强阳性表达。相对于用药3天,参麦+丹参组在7天时bFGF mRNA基因表达明显降低。
实验研究证实:活血、益气、清热法具有确切地改善创伤修复的作用,其中碱性成纤维细胞生长因子(basic fibroblast growth factor,bFGF)mRNA表达和创面修复早、中期肉芽组织中Ⅰ、Ⅲ型胶原的变化可以部分解释其促进创伤修复的机理。
讨论
综合临床研究和实验研究结果,可以得出以下结论:清热、活血、益气法能够促进创伤修复;活血法在治疗创伤中起到重要作用,可全程使用,有降低瘢痕生成的可能;清热法对创伤早期的修复意义较大;益气法可促进创面的肉芽生长,使创面尽早愈合,并且可以提高愈合质量;在围手术期可运用分期辨证理念指导术后康复;在创伤修复领域,中医药有独特的临床优势,其机理有待进一步深入研究。
Perioperation stage is one of the hot issues in surgery,which is increasingly considered.The purpose of repair in trauma is changing from speed to quality in healing.The connotation of repair in trauma is expanding expressed with the study from the body surface to inside,from local area to all over the body,from macroscopic to microcosmic.It is believed by the modern medicine:there are not only changes in morphology but also in biochemics in repair in trauma.In the later one,growth factor and collagen such as FGF, typeⅠ,Ⅲcollagen play important roles during the repair.
In traditional Chinese medicine(TCM),it is thought that the forein coup result in Qi and blood stagnation,stagnation of Yingqi,meridian blocking, disorder between viscera and organs after trauma which lead to heat,stagnated blood,deficency of healthy Qi and so on consequently.Reparative process after surgical trauma showed obvious different properties in the different stages. During the early stage,the patients are mostly manifested with pyretic syndrome and syndrome of blood stasis;during the intermediate stage,they are mostly manifested with deficient syndrome and syndrome of blood stasis; during the stage of postoperative rehabilitation,they are always with deficient syndrome.The previous studies indicated that the traditional Chinese medicine can produce marked effects through multiple targets and links during repair in trauma,while the effects and mechanisms of TCM are not clear in the process of the wound repair yet.
With the guidance of the TCM theory,this study considered the regularity of wound repair in toto;analysized and summarized the effects of promoting blood flow,tonifying Qi,clearing away heat on the different stages in wound repair in clinic.On the other hand,the basic fibroblast growth factor(bFGF) and typeⅠ,Ⅲcollagen in granulation tissues were detected in the early stage during wound repair in rat,in order to observe the effects of the treatment of promoting blood flow,tonifying Qi,clearing away heat on the wound repair in rat and to approach the mechanisms.
Part one:clinical study
Patients after open cystectomy were selected and divided into 2 groups in random.There are no obvious differences between groups in the sex,age, operation time,anaesth time,volume of bleeding,bile bacteria culture.The control group was treated with common practice;the treatment group was treated according to the rules of TCM above:in addition to common practice,the patients were i.v administrated with Qingkailing injection(to clear away the heat)and Danshen injection(to promoting the blood flow)at the operation day and 1-3 days after operation,with Shenmailing injection(to tonify the Qi)and Danshen injection at the 4-7 days after operation.The body temperature recovery time,wound repair information,white blood cells(WBC)recovery time, the recovery time of gastrointestinal functions,body weigh,complications, length of stay in hospital,cost of hospitalization were observed and investigated.It was found that the treatment group was better than the control groups in the following items such as body temperature recovery time,WBC recovery time,time of passage of gas by anus,first defecation time after operation,the time to take out stitches,body weight lessening and so on (P＜0.05);the incidence of complications(pulmonary infection,urinary system infection,split of the infected wound)in treatment group was lower than control group.The treatment group did not add the cost of hospitalization compared with the control group.
Part two:experimental study
SD rats were divided to several groups:model group,clearing away heat +promoting the blood flow treatment group,tonifying Qi+promoting the blood flow treatment group.Rats in each group were killed at day 3 or 7 to take the neogenetic granulation tissue on the wound surface to observe bFGF mRNA expression by quantities PCR,pathological changes by HE stain,typeⅠ,Ⅲcollagen production by immunohistochemistry methods.The basic information such as food-intake,loss of body weight was also observed after operation.
The results showed that the food-intake and the loss of body weight were improved from day 1-3 after operation in Danshen+Qingkailing group compared with model group;improved from day 4-7 in Danshen+Shenmai group compared with model group.
In Qingkailing +Danshen group,it was seen that the granulation tissue grew red and the wound surface diminished obviously at day 3.In addition, the blood scabs formation with oozy fluid and inflammation cells under it, repair hyperplasia could beobserved by HE stain;immunohistochemistry results showed that the low to medium amounts of typeⅠcollagen expressed and low amount of typeⅢcollagen expressed in this group;the bFGF mRNA expression increased compared with the model group.At day 7,the food-intake and the activity gradually recovered;the granulation tissues proliferated with afferent,clumping and neoplastic characters which covered the wound surface completely and the tissue was not smooth.In HE stain,there were still blood scabs formation under which small amounts of oozy fluid and inflammation cells were seen,the repair hyperplasia was obvious.TypeⅠ,Ⅲcollagen expression were similar to day 3,while the bFGF mRNA increased compared with day 3.
At day 3 post operation in Shenmai+Danshen group,it was seen that the granulation tissue grew evenly and the wound surface diminished obviously. In HE stain,the granulation tissue hyperplasia with more inflammatory cell infiltration;immunohistochemistry results showed there are weak expression of typeⅠ,Ⅲcollagen in this group;the bFGF mRNA expression increased compared with the model group.At day 7,the neoplastic granulation tissues were smooth and part of the wound had been repaired.In HE stain,there were many neoplastic granulation tissues which was similar to day 3.The TypeⅠ,Ⅲcollagen expressions were stronger than day 3,while the bFGF mRNA reduced obviously compared with day 3.
This study partly confirmed that to prompt the blood flow,tonify Qi,clear away heat could improve would repair,which may partly due to the effects on the bFGF and typeⅠ,Ⅲcollagen production in the early and intermediate stage during wound repair.
Discussions:
It was concluded that to prompt the blood flow,tonify Qi,clear away heat could improve would repair according to the results in clinical study and experimental study.To prompt the blood flow played important rules in the treatment of wound which may reduce the scar formation and could be used in the whole stage;To clear away heat was more important at the early stage; to tonify Qi could prompt the growth of the granulation tissues which may lead to repair the wound as early as possible and improve the healing quality.To differentiation of symptoms and signs according to the different stage in per operation stage could guide on the recovery postoperation.The TCM showed advantages in wound repair while the mechanisms need ho be further investigated.
中医认为:创伤打击致使机体气血凝滞、营气不从,经络阻塞,脏腑失和;导致郁而化热、瘀血停滞、正气亏虚等。机体修复过程有明显的时段性,早期患者多表现为热证和血瘀证,中期患者多表现为虚证和血瘀证,术后康复期多表现为虚证。中药可以多靶点、多环节地发挥作用。近年有关创伤修复的逐步展开,但对中医药在创伤修复过程中的作用规律及机理仍缺乏深入研究。
本研究试图在中医理论指导下,整体思考创伤修复的变化规律,根据既往的临床实践,通过临床研究,分析活血、益气、清热法对创伤组织修复不同阶段的的作用。结合临床资料总结,探讨围手术期不同时段的中医辩证用药的共性及其优势。并通过创伤修复实验研究,检测碱性成纤维细胞生长因子(basic fibroblast growth factor,bFGF)mRNA表达的关系、观察各组大鼠创面修复早期肉芽组织中Ⅰ、Ⅲ型胶原的变化;来探讨清热、活血、益气法改善大鼠伤口愈合的作用,分析其促进创面愈合的实际作用与机理。
临床研究
选择开放胆囊切除术的病例,随机分组。两组病人在性别、年龄、手术时间、麻醉时间、出血量、胆汁细菌培养等方面比较,P均＞0.05,差异无统计学意义,具有可比性。对照组按照常规临床路径治疗;治疗组方案在标准临床路径的基础上加用分期中医辩证处方治疗方案:手术当日及术后第1-3天静滴清开灵20ml+生理盐水100ml,丹参针20ml+生理盐水100ml;术后第4-7天静滴参麦针20ml+生理盐水100ml,丹参针20ml+生理盐水100ml。观察患者术后体温恢复正常时间、伤口愈合情况、白细胞恢复正常时间,胃肠功能恢复时间、体重变化、伤口愈合情况、胃肠道功能回复情况、并发症情况、住院时间、住院费用等指标。
临床研究结果:治疗组在体温恢复正常时间、血WBC恢复正常时间、肛门排气时间、术后首次排便时间、拆线时间、体重减轻等方面,均优于对照组(P＜0.05);治疗组并发症(肺部感染、泌尿系感染、伤口感染裂开)发生率低于对照组;治疗组并不增加患者住院费用。
实验研究
实验动物及分组SPF级SD大鼠,雄性,随机分组,创面造模后喂药分第3天、第7天两个观测时段。每个时段又分为模型组(生理盐水)、清热+活血组、益气+活血组。根据成人日服用药量的5倍计算大鼠腹腔注射剂量,用药后第3天、7天(每组8-13号动物),取新生创面肉芽组织,送检。
基本观察:术后丹参+清开灵组在造模后1—3天进食量、体重减轻优于模型组;丹参+参麦灵组在造模后4—7天进食量、体重增加优于模型组(P＜0.05)。
实验结果(以下P＜0.05或P＜0.01)):
清开灵+丹参组用药3天后,肉芽生长明显红活,创面缩小程度显著;HE染色表现:镜下见创伤表面血痂形成,痂下可见渗出的液体及炎症细胞(图5),各例组织均可见肉芽组织修复性增生的改变(图6)。免疫组化:镜下见各例组织中均有少量至中等量黄褐色Ⅰ型胶原阳性表达。镜下见各例组织中均有少量黄褐色Ⅲ型胶原弱阳性表达;bFGF mRNA表达比造模组增加显著。七天后表现为:进食、活动等逐渐恢复,丹参+清开灵组肉芽组织明显向心性、团块状、瘤性增生,创面由于为来自不同方向生长肉芽组织而体积变大,将创面完全封闭,但组织不平整;HE染色表现:镜下仍可见创伤表面血痂形成,痂下少量渗出的液体及炎症细胞(图7),肉芽组织修复性增生明显(图8)。免疫组化:镜下所见与3天时相似,少至中等量Ⅰ型胶原阳性表达。镜下所见与3天时相似,少量Ⅲ型胶原弱阳性表达,部分组织内有少量黄褐色Ⅲ型胶原弱阳性表达。相对于用药3天,清开灵+丹参组在7天时bFGF mRNA基因表达明显增加。
参麦+丹参组用药3天后,肉芽生长较均匀,创面缩小;HE染色表现:镜下见创面肉芽组织增生伴较多炎细胞浸润(图9)。免疫组化:镜下见各例组织中均有黄褐色Ⅰ型胶原弱阳性表达。少量黄褐色Ⅲ型胶原弱阳性表达。bFGF mRNA表达明显增加。七天后,增生出的肉芽组织明显平整,部分增生组织平整创口处被修复;HE染色表现:镜下所见与3天相似,镜下可见较多肉芽组织增生;免疫组化:镜下见各例组织中Ⅰ型胶原表达较3天时明显,个别组织出现Ⅰ型胶原强阳性表达。镜下见各例组织中Ⅲ型胶原表达较3天时明显增多,但未见强阳性表达。相对于用药3天,参麦+丹参组在7天时bFGF mRNA基因表达明显降低。
实验研究证实:活血、益气、清热法具有确切地改善创伤修复的作用,其中碱性成纤维细胞生长因子(basic fibroblast growth factor,bFGF)mRNA表达和创面修复早、中期肉芽组织中Ⅰ、Ⅲ型胶原的变化可以部分解释其促进创伤修复的机理。
讨论
综合临床研究和实验研究结果,可以得出以下结论:清热、活血、益气法能够促进创伤修复;活血法在治疗创伤中起到重要作用,可全程使用,有降低瘢痕生成的可能;清热法对创伤早期的修复意义较大;益气法可促进创面的肉芽生长,使创面尽早愈合,并且可以提高愈合质量;在围手术期可运用分期辨证理念指导术后康复;在创伤修复领域,中医药有独特的临床优势,其机理有待进一步深入研究。
Perioperation stage is one of the hot issues in surgery,which is increasingly considered.The purpose of repair in trauma is changing from speed to quality in healing.The connotation of repair in trauma is expanding expressed with the study from the body surface to inside,from local area to all over the body,from macroscopic to microcosmic.It is believed by the modern medicine:there are not only changes in morphology but also in biochemics in repair in trauma.In the later one,growth factor and collagen such as FGF, typeⅠ,Ⅲcollagen play important roles during the repair.
In traditional Chinese medicine(TCM),it is thought that the forein coup result in Qi and blood stagnation,stagnation of Yingqi,meridian blocking, disorder between viscera and organs after trauma which lead to heat,stagnated blood,deficency of healthy Qi and so on consequently.Reparative process after surgical trauma showed obvious different properties in the different stages. During the early stage,the patients are mostly manifested with pyretic syndrome and syndrome of blood stasis;during the intermediate stage,they are mostly manifested with deficient syndrome and syndrome of blood stasis; during the stage of postoperative rehabilitation,they are always with deficient syndrome.The previous studies indicated that the traditional Chinese medicine can produce marked effects through multiple targets and links during repair in trauma,while the effects and mechanisms of TCM are not clear in the process of the wound repair yet.
With the guidance of the TCM theory,this study considered the regularity of wound repair in toto;analysized and summarized the effects of promoting blood flow,tonifying Qi,clearing away heat on the different stages in wound repair in clinic.On the other hand,the basic fibroblast growth factor(bFGF) and typeⅠ,Ⅲcollagen in granulation tissues were detected in the early stage during wound repair in rat,in order to observe the effects of the treatment of promoting blood flow,tonifying Qi,clearing away heat on the wound repair in rat and to approach the mechanisms.
Part one:clinical study
Patients after open cystectomy were selected and divided into 2 groups in random.There are no obvious differences between groups in the sex,age, operation time,anaesth time,volume of bleeding,bile bacteria culture.The control group was treated with common practice;the treatment group was treated according to the rules of TCM above:in addition to common practice,the patients were i.v administrated with Qingkailing injection(to clear away the heat)and Danshen injection(to promoting the blood flow)at the operation day and 1-3 days after operation,with Shenmailing injection(to tonify the Qi)and Danshen injection at the 4-7 days after operation.The body temperature recovery time,wound repair information,white blood cells(WBC)recovery time, the recovery time of gastrointestinal functions,body weigh,complications, length of stay in hospital,cost of hospitalization were observed and investigated.It was found that the treatment group was better than the control groups in the following items such as body temperature recovery time,WBC recovery time,time of passage of gas by anus,first defecation time after operation,the time to take out stitches,body weight lessening and so on (P＜0.05);the incidence of complications(pulmonary infection,urinary system infection,split of the infected wound)in treatment group was lower than control group.The treatment group did not add the cost of hospitalization compared with the control group.
Part two:experimental study
SD rats were divided to several groups:model group,clearing away heat +promoting the blood flow treatment group,tonifying Qi+promoting the blood flow treatment group.Rats in each group were killed at day 3 or 7 to take the neogenetic granulation tissue on the wound surface to observe bFGF mRNA expression by quantities PCR,pathological changes by HE stain,typeⅠ,Ⅲcollagen production by immunohistochemistry methods.The basic information such as food-intake,loss of body weight was also observed after operation.
The results showed that the food-intake and the loss of body weight were improved from day 1-3 after operation in Danshen+Qingkailing group compared with model group;improved from day 4-7 in Danshen+Shenmai group compared with model group.
In Qingkailing +Danshen group,it was seen that the granulation tissue grew red and the wound surface diminished obviously at day 3.In addition, the blood scabs formation with oozy fluid and inflammation cells under it, repair hyperplasia could beobserved by HE stain;immunohistochemistry results showed that the low to medium amounts of typeⅠcollagen expressed and low amount of typeⅢcollagen expressed in this group;the bFGF mRNA expression increased compared with the model group.At day 7,the food-intake and the activity gradually recovered;the granulation tissues proliferated with afferent,clumping and neoplastic characters which covered the wound surface completely and the tissue was not smooth.In HE stain,there were still blood scabs formation under which small amounts of oozy fluid and inflammation cells were seen,the repair hyperplasia was obvious.TypeⅠ,Ⅲcollagen expression were similar to day 3,while the bFGF mRNA increased compared with day 3.
At day 3 post operation in Shenmai+Danshen group,it was seen that the granulation tissue grew evenly and the wound surface diminished obviously. In HE stain,the granulation tissue hyperplasia with more inflammatory cell infiltration;immunohistochemistry results showed there are weak expression of typeⅠ,Ⅲcollagen in this group;the bFGF mRNA expression increased compared with the model group.At day 7,the neoplastic granulation tissues were smooth and part of the wound had been repaired.In HE stain,there were many neoplastic granulation tissues which was similar to day 3.The TypeⅠ,Ⅲcollagen expressions were stronger than day 3,while the bFGF mRNA reduced obviously compared with day 3.
This study partly confirmed that to prompt the blood flow,tonify Qi,clear away heat could improve would repair,which may partly due to the effects on the bFGF and typeⅠ,Ⅲcollagen production in the early and intermediate stage during wound repair.
Discussions:
It was concluded that to prompt the blood flow,tonify Qi,clear away heat could improve would repair according to the results in clinical study and experimental study.To prompt the blood flow played important rules in the treatment of wound which may reduce the scar formation and could be used in the whole stage;To clear away heat was more important at the early stage; to tonify Qi could prompt the growth of the granulation tissues which may lead to repair the wound as early as possible and improve the healing quality.To differentiation of symptoms and signs according to the different stage in per operation stage could guide on the recovery postoperation.The TCM showed advantages in wound repair while the mechanisms need ho be further investigated.
引文
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