摘要
1研究目的
急性颅脑损伤已成为一种严重危害人类健康的疾病。颅脑继发性损伤是导致颅脑损伤病人死亡或残废的重要原因。继发性颅脑损伤发生机制十分复杂,其中创伤性脑缺血是目前研究的一个难点和热点。在防治颅脑损伤后创伤性脑缺血方面许多学者做出了很大的努力,逐渐形成了一套治疗方案,有控制血压、保持呼吸通畅、适时手术、高压氧治疗、亚低温治疗、使用促进脑细胞代谢药物等,这些措施都能在一定程度上改善创伤性脑缺血状态以及预后,但各自有其不足之处,许多方面还存在争议,有待进一步深入验证。
文献调研和临床实践证实,中西医结合在防治重型颅脑损伤方面能发挥显著疗效,然而其具体的作用机理,特别是对创伤性脑缺血的防治作用机理研究报道甚少,阻碍了临床拓展运用。我科在此领域经过多年临床应用和经验积累,认为在西医常规治疗基础上,结合运用活血化瘀、豁痰开窍及通腑泻热中药,治疗重型颅脑损伤能祛瘀血、豁痰湿、泻内热、起到开窍醒脑,改善缺血缺氧状态,有效解决颅脑外伤后出现的神昏、高热、呼吸困难、肢体功能障碍、大便闭结、痰多粘稠等症状,较好地改善了预后。因此,理论上讲,在西医常规治疗基础上,结合运用活血化瘀、豁痰开窍及通腑泻热中药对于防治重型颅脑损伤后创伤性脑缺血应该有确切的作用机制和明确的临床疗效,值得我们研究证实。
本研究拟从临床研究和实验研究两方面着手,就中西医结合对重型颅脑损伤后创伤性脑缺血的防治作用机理进行深入研究,为拓展其临床运用提供有价值的依据。
2研究方法与结果
2.1中西医结合对重型颅脑损伤后创伤性脑缺血防治作用机理的临床研究
方法:我们选择自2007年1月至2009年3月在广州中医药大学第一附属医院颅脑科病房确诊的急性重型颅脑损伤住院病人80例,严格执行纳入标准和排除标准,随机分组为对照组和治疗组,从入院第1天开始,采用不同治疗处理方案,对照组采用单纯西医常规治疗,治疗组则在西医常规治疗基础上加用活血化瘀汤药鼻饲、豁痰开窍中药针剂静滴、配合通腑泻热中药灌肠治疗,从脑血管痉挛发生率、颅内血流速度变化、GCS计分变化、颅脑CT检查外伤性脑梗塞的发生率、相关安全性指标检测等方面进行连续动态观察,持续18天。数据处理,采用SPSS10.0软件包分析,计量资料用t检验,计数资料用X~2检验,显著性水平取α=0.05。
结果:(1)入院后第3天两组患者MCA、ACA血流速度的均数有显著性差异(p＜0.05);第6天两组患者MCA、ACA、PCA、VA血流速度的均数有显著性差异(P＜0.05),其中以MCA流速的均数相差最大:第12天两组患者MCA、ACA、BA、VA血流速度的均数有显著性差异(P＜0.05);第18天两组患者MCA、PCA的血流速度的均数有统计学差异(P＜0.05),但差异均较小。(2)入院后第3天、第6天、第12天两组患者脑血管痉挛发生率均存在显著性差异(P＜0.05),两组血管痉挛情况随着时间延长均逐渐缓解;(3)两组患者GCS计分在入院第1天无显著性差异(P＞0.05),第3天、第6天、第12天、第18天两组患者的GCS计分均存在显著性差异(P＜0.05);(4)两组患者创伤性脑梗塞发生率分别为30.0%和10.0%,存在显著性差异(P＜0.05);(5)在治疗第6天、第12天复查血分析、肝肾功能,结果提示在用药过程中无明显毒副作用。
2.2补阳还五汤促进血管内皮细胞增殖及血管新生的实验研究
2.2.1补阳还五汤含药血清的制备
方法:成年新西兰兔,雌雄各半,分高、中、低剂量中药组和生理盐水对照组,每天灌胃一次,连续3天采血。麻醉后无菌操作下右颈动脉采血,离心、收集血清、灭活、过滤,分组包装,低温保存。
结果:本法采血前饲养动物情况良好,右颈动脉处采血每只兔可收集50ml左右新鲜血液,操作容易,采血量较多。
2.2.2兔血管内皮细胞的体外原代培养与传代
方法:取新西兰兔胸主动脉,外翻置于0.1%Ⅱ型胶原酶中消化收集消化液,离心收集细胞分瓶培养,72小时后换液培养;原代细胞8天左右铺满瓶底,用0.25%的胰蛋白酶消化,吹打分散,计数,分瓶传代培养。
结果:原代内皮细胞48小时大部分贴壁,细胞呈小圆形、多角形,有椭圆形核,72小时伪足伸出;4-8天内细胞迅速生长,8-9天长成单层,铺满瓶底。传代细胞4—8小时内贴壁伸出伪足,7天内长成单层;10天以上呈鹅卵石样紧密排列,可见旋涡状;内皮细胞传10代以上其特征不变。
2.2.3兔血管内皮细胞的鉴定试验
方法:倒置相差显微镜下观察细胞分裂形成单层时的形态进行鉴定;免疫荧光法鉴定:将细胞培养在载玻片上形成单层贴片,经酒精、ⅧR—Ag、羊抗鼠IgG及荧光标记的SABC作用,放于荧光显微镜下观察,并作阴性对照。
结果:内皮细胞呈小圆形、多角形,有椭圆形核。免疫荧光法可见大量的绿色细胞,细胞浆、膜上有明显染色,细胞核无染色。
2.2.4兔血管内皮细胞的增殖及药物血清的作用
方法:用MTT法测细胞增殖,绘制细胞生长曲线;将不同剂量不同浓度药物血清作用促进细胞增殖,比较不同剂量不同浓度药物血清的作用效果。
结果:5—8天为快速增长期,10天左右达到增殖的高峰,11—14天后增殖呈平坦趋势。相同剂量不同浓度中10%含药血清组、20%含药血清组较5%的含药血清组有统计学差异(P＜0.05);相同浓度不同剂量中剂量组与低剂量组,高剂量组与低剂量组间有统计学差异(P＜0.05)。
2.2.5兔血管内皮细胞分裂指数的测定
方法:采用Giemsa染色法,先于低倍光镜下选择分散较好、细胞核着色清楚而胞质无色的细胞,再转到高倍镜下观察细胞是否处于分裂期;每个样本计数1000个以上的细胞,记录分裂期细胞数和间期细胞数,再计算细胞分裂指数。
结果:细胞在培养阶段中80%以上的细胞处于细胞间期,20%以下的细胞处于分裂期。不同含药血清培养的内皮细胞均较空白组细胞的分裂指数增加,增加幅度组间有差异;各组6-8天达到分裂高峰,以后分裂指数逐步下降,11-12天降至1%左右。
2.2.6兔血管内皮细胞的透射电镜观察
方法:用不同剂量含药血清培养干预细胞后,准备细胞样品,用JEM-200CX透射电镜观察内皮细胞形态,包括细胞核、细胞器、细胞浆、细胞内脂滴大小分布等。
结果:含药血清细胞较空白组细胞排列明显紧密,细胞核较大,染色质清晰;胞质内粗面内质网增生扩张;线粒体相对发达,嵴结构及嵴上颗粒清晰;亦有大量吞饮小泡,细胞间有内皮细胞间连接或桥粒,可见细胞内有大糖原颗粒和细胞特殊颗粒等。
2.2.7补阳还五汤含药血清促进鸡胚绒毛尿囊膜血管生成的试验
方法:将高、中、低剂量中药含药血清及空白对照血清作为被测物置于鸡胚绒毛尿囊膜表面的载体上,作用充分后后制备CAM标本,解剖显微镜下观察、计算机图像分析仪图像分析,得到相关定量数据,进一步统计学分析。
结果:含药血清具有明显的促进鸡胚绒毛尿囊膜血管生成的作用,用药组较空白组有显著性差异。
3结论
3.1重型颅脑损伤后创伤性脑缺血的中医病机可概括为气滞血瘀,痰瘀热交结,闭阻清窍。
3.2在西医治疗基础上结合活血化瘀汤药鼻饲、豁痰开窍针剂静滴及通腑泻热中药灌肠,能降低脑外伤后颅内血流速度,防治脑血管痉挛,减少脑梗塞发生率,改善脑缺血状态,提高GCS评分,改善预后,且对患者无明显肝肾功能损害,安全有效。
3.3补阳还五汤可能通过改善血管内皮细胞能量合成,促进细胞新陈代谢及功能活动,诱导产生促进内皮细胞增殖的生长因子来促进血管内皮细胞增殖;可能激活血管内皮及外膜细胞,促进其分裂、出芽和迁移,启动血管新生程序,促进内皮细胞分化及细胞连接,形成原始血管腔,并进一步促进新生血管稳定和成熟来促进血管新生。
3.4综合临床和实验研究:中西医结合对重型颅脑损伤后创伤性脑缺血有显著的防治效果,其作用机理之一,可能是通过保护受损脑微血管内皮细胞,促进内皮细胞增殖和迁移,修复受损微血管,并在缺血区域促进生成新血管,提高局部脑血流,改善脑微循环,从而保护脑组织和神经功能。
4.本课题的特点和创新性
4.1在西医常规治疗基础上运用活血化瘀、豁痰开窍及通腑泻热中药联合用药防治重型颅脑损伤后创伤性脑缺血是一个治疗理念的创新。
4.2对重型颅脑损伤后创伤性脑缺血的病机认识,我们第一次完整概括为气滞血瘀,痰瘀热交结,闭阻清窍。
4.3将中药血清药理学、血管内皮细胞培养及鸡胚绒毛尿囊膜血管生成实验等相结合,应用到中医药防治创伤性脑缺血机理的研究,是一个全新的尝试。
1 Objective of research
Acute brain injury has become a serious hazard to human health.Secondary brain injury with traumatic brain injury is the important reason leading to patients' death or disability.Occurrence mechanism of secondary brain injury is very complex,which ischemic trauma is a difficult and hot point in the current study.
In the prevention and treatment of cerebral ischemia after traumatic brain injury,many scholars have made great efforts.Now has a set of programs in modern medical,including control of blood pressure,keeping breathing smooth, timely surgery,hyperbaric oxygen therapy,mild hypothermia,the use of drugs to promote brain cell metabolism,etc.To some extent these measures can improve the state of traumatic cerebral ischemia and prognosis,but have their own inadequacies.There are still many aspects of the dispute,subject to further verification.
Literature research and clinical practice suggested Traditional Chinese and western medicine in the prevention and treatment of severe head injury play a significant efficacy,and application have been gradually extended. But the studies of specific mechanism for prevention and treatment of traumatic ischemic have reported much less,hindering the development of clinical use.
In this area,our department has many years of clinical experience from conventional therapy in Traditional Chinese and Western Medicine.Based on conventionality therapy in Western medicine,a combination therapy of Huoxuehuayu,Huotankaiqiao,Tongfuxiere Chinese medicine has been used for treatment severe brain injury.It can remove static blood,resolve excrescently phlegm,promote diarrhea heat,speed awake,improve the status of ischemia and hypoxia.It can solve exanimation after brain injury,high fever,difficulty in breathing,physical dysfunction,stool closed end.It also can resolve symptoms such as viscous phlegm excrescently.So the better prognosis can be improved.Therefore,in theory,based on conventionality therapy in Western medicine,the combination therapy of Huoxuehuayu, HuotanKaiqiao,Tongfuxiere Chinese medicine for prevention and treatment of traumatic brain ischemia after severe head injury should be have some exact mechanisms and clear clinical efficacy,worthy of our study.
From two aspects of the clinical research and experimental study,we use Traditional Chinese and Western Medicine for prevention andtreatment cerebral ischemia in severe traumatic brain injury,that will deep research of the mechanism and provide value evidence for expanding its clinical use.
2 Research Methods and Results
2.1 The clinical study on Traditional Chinese and Western Medicine prevention and control mechanism of cerebral ischemia in the severe traumatic brain injury.
Methods:We selected 80 cases that patients were confirmed with acute severe head injury in the neurosurgery department of the First Affiliated Hospital of Guangzhou University of Traditional Chinese Medicine from January 2007 to March 2009.We strictly performed the inclusion criteria and exclusion criteria.These patients were randomized for the control group and treatment group.From the first day in hospital,a different treatment optionhas been done,the control group being done only by a simple conventional western medicine treatment,the treatment group being done in the conventional Western medicine combined with treatment with nasal feeding on the Huoxuehuayu decoction,with intravenous infusion of Huotankaiqiao injection of Chinese medicine and with enema Tongfuxiere of the Traditional Chinese medicine.From the incidence of cerebral vasospasm,intracranial blood flow velocity changes, GCS score changes,the incidence of cerebral infarction in traumatic brain by CT checking,the security aspects of monitoring,indicators dynamic observation of continuous,the study sustained for 18 days.Data processing has been done as analysis of the use of SPSSIO.O packages,metrology data using "t" test,"X~2" test with count data,check the level of significantα= 0.05.
Results:(1)Admitted to hospital the third day two groups of patients with MCA,ACA mean blood flow velocity were significantly different(P＜0.05); The sixth day,two groups of patients with MCA,ACA,PCA,VA blood flow velocity were significantly different(P＜0.05),of which the mean MCA flow velocity difference was the largest;The twelfth day of the two groups of patients with MCA,ACA,BA,VA mean blood flow velocity were significantly different(P＜0.05);The 18th day of the two groups of patients with MCA,PCA of the mean blood flow velocity was significant difference(P＜0.05),but the difference was smaller.(2) The third day after admission,the sixth day,the twelfth day of two groups of patients with the incidence of cerebral vasospasm were significantly different(P＜0.05),two cases of vasospasm were gradually extended over time to ease;(3)Two groups of patients with GCS score on the first day was not significantly different(P＞0.05),the third day,the sixth day,the twelfth day,the 18th day of two groups of patients with GCS score were significantly different(P＜0.05);(4) Two groups of patients with traumatic cerebral infarction rates were 30.0%and 10.0%,there was significantly different(P＜0.05);(5) In the treatment of the sixth day,the twelfth day to review blood analysis and liver and kidney function examinatin, results suggested that in the course of medication there was no significant side effects.
2.2 The experimental research of Buyanghuanwu decoction to promote vascular endothelial cell proliferation and angiogenesis.
2.2.1 Preparation of serum containing Buyanghuanwu decoction
Methods:Between male and female,adult New Zealand rabbits were equally divided into high,mediumand low-dose medicine group and the saline control group,administered once daily for 3 consecutive days.Asepsis under anesthesia,rabbit blood form the right carotid artery was collected and centrifuged.The serum was collected,inactivated,filtered,group packaged, cold storaged.
Results:All of animals kept in good condition beforeblood collection. About 50ml of fresh blood could be collected from the right carotid artery of each rabbit.This method was easy and reliable.
2.2.2 Rabbit vascular endothelial cells in vitro primary culture and subculture
Methods:The eversion New Zeal rabbit thoracic aorta was put at 0.1%Ⅱ-type collagenase to digest.The digestive juice was collected and centrifuged.The cells were collected and put into bottles for culture.After 72 hours the foster liquid was changed.After 8 days primary cells covered around the bottom.Then 0.25%steapsin was put into the flask to digest for a few minutes.Cells were dispersed wind,counted,cultured in different bottle.
Results:The primary endothelial cells need 48 hours or so to become the majority adherent cells.Cells were small round,polygonal,and oval-shaped core.After 72 hours the cells extended pseudopodia;4-8 days cells grew rapidly.8-9 days cells grew in primary monolayer,covered the bottom.The Subcultured endothelial cells adhered in 4-8 hours and extended out pseudopodia,7 days cells grew in primary monolayer,more than 10 days cells were closed with cobblestone-like,non-overlapping,we can see the vortex-like cells;Endothelial cells kept the same characteristic more than 10 generation.
2.2.3 Vascular endothelial cells of rabbit test
Methods:Morphological identification was done under inverted phase contrast microscope through observation of cell division to form single-layer forms.Immunofluorescence identification was done.Firstly the cells were cultured in glass slide to form a single-chip,then which were processed by alcohol,ⅧR-Ag,sheep anti-mouse IgG,the SABC and fluorescent-labeled. At last that were put in fluorescent microscopy to observe,negative controlled.
Results:The endothelial cells were small round,polygonal,and oval-shaped nuclear.Immunofluorescence shows that a large number of green cells,plasma cells,a clear membrane staining,non-staining nuclei.
2.2.4 Rabbit vascular endothelial cell proliferation and the role of serum
Methods:We used measurement of cell proliferation MTT and cell growth curve mapping for testing the role of different doses of the same concentrations of drug serum to promote cell proliferation,testing the role of different concentrations of the same doses of drug serum to promote cell proliferation.
Results:5-8 days was a period of rapid growth,about the tenth day cells reach the peak of cell proliferation,after 14 days the trend of cell proliferation was flat.The same dose of different concentrations of serum containing 10%group and 20%group were significantly different than the serum containing 5%group(P <0.05).The same concentration of different doses of serum in medium-dose group and high-dose group were significantly different than the low-dose group(P <0.05).
2.2.5 Rabbit vascular endothelial mitotic index determination
Methods:By Giemsa staining,firstly in low magnification light microscopy we choiced scattered better cells which the nucleus and cytoplasm coloring were clear,and then transferred to high-power microscope to observe cells division period.Each sample was counted for more than 1000.By recording the number of split-phase cells and the number of interphase cells,the cells mitotic index was calculated.
Results:In the whole phase of the cells cultured,more than 80%of the cells were in interphase,20%of the cells were in divided following period. Different drug-containing serum culture of endothelial cells than those in the blank cells can increase the mitotic index.In each group 6-8 days cells reached the peak mitotic and then gradually decreased,after 11-12 days reduced to 1%.
2.2.6 Observation Rabbit vascular endothelial cells in TEM
Methods:Have been intervened with the serum containing different doses of drug,cell samples were prepared.By JEM-200CX transmission electron microscope,endothelial cells were observed including cells within the nucleus,organelle,cytoplasm,cell size distribution of lipid droplets.
Results:The cells which were intervened with the serum containing different doses of drug in each group were significantly closer with larger nuclei,clearer chromatin than the blank cells;Intervened with the serum containing drug,the cells had cytoplasm with rough endoplasmic reticulum expansion hyperplasia,mitochondrial relatively well-developed ridge crest on the particle structure.There was a substantial amount pinocytotic vesicles of the cells between the endothelial cells or between connecting desmosomes. These cells can be seen with large glycogen granules and specific granules.
2.2.7 Serum containing Buyanghuanwu decoction to promote the chick embryo chorioallantoic membrane angiogenesis test
Methods:The serum containing high,medium and low doses of medicine and the blank control serum were placed in the carrier on the surface of the chick embryo chorioallantoic membrane to be tested.When the specimens of CAM were prepared,anatomical observation under a microscope and computer image analysis image analysis have been related to quantitative data for further statistical analysis.
Results:The serum containing drug can significantly promote chick embryo chorioallantoic membrane angiogenesis.
3 Conclusion
3.1 The pathology in TCM about cerebral ischemia in the severe traumatic brain injury can be summarized as Qizhixueyu,then phlegm,blood stasis and thermal cross-noded,mind closed.
3.2 Based on conventionality therapy in Western Medicine,the combination therapy of Huoxuehuayu,HuotanKaiqiao,Tongfuxiere Chinese medicine for prevention and treatment of traumatic brain ischemia after severe head injury, that can reduce post-traumatic intracranial cerebral blood flow velocity, prevent and treat with cerebral vasospasm and reduce cerebral infarction the incidence,improve the state of cerebral ischemia,improve patients with GCS score and the prognosis.The liver and kidney function examination in patients have not benen obviously damaged.This combination therapy was safe and effective.
3.3 Buyanghuanwu Decoction could improve the vascular endothelial cells energy synthesis,promote cell metabolism with activity function,induce to secrete the growth factor to promote endothelial cells proliferation.
3.4 Buyanghuanwu Decoction may also be activated to the outer membrane of vascular endothelial cells,and promote their division,budding and migration. And activate angiogenesis start procedures,promote endothelial cell differentiation and cell connections to form a primitive lumen,and further promote the stability and neovascularization mature to promote angiogenesis.
3.5 Integrated Clinical and Experimental Research:Using Traditional Chinese and Western Medicine for cerebral ischemia in severe traumatic brain injury, that was significant effect,one of its mechanism may be sure by protecting brain microvascular endothelial cells,promoting endothelial cell proliferation and migration,repairing the damaged microvascular,promoting ischemic regions to generate new blood vessels,improving regional cerebral blood flow,improving cerebral microcirculation.Thereby this therapy can protect brain tissue and nerve function.
4.The features and innovative of this subject
4.1 Based on conventionality therapy in Western medicine,a combination therapy of Huoxuehuayu,Huotankaiqiao,Tongfuxiere Chinese medicine has been used for treatment with severe brain injury,which is a innovation concept of treatment.
4.2 we firstly summed up the TCM pathogenesis of Cerebral Ischemia in severe traumatic brain injury,which is Qizhixueyu,phlegm and blood stasis and thermal cross-knot resistance then mind being closed.
4.3 By using of technology of serum to Chinese pharmacology,vascular endothelial cell culture and chick embryo chorioallantoic membrane angiogenesis experiments,we studied on mechanism of Traditional Chinese Medicine prevention and treatment of Cerebral Ischemia in the severe traumatic brain injury.This is a new attempt.
引文
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