| Objective: China is a high-incidence country of esophageal squmous cancer(ESC), more than 50% of all esophageal cancer patients in the world are Chinese, the five-year survival rate of this disease on the whole was less than 10%, the human being are threatened by ESC greatly. So ESC is the disease to be controlled and prevented in our country mainly. But the authentic factors which caused ESC is still unknown, it is difficult to prevent this disease occurring etiologically, so early diagnosis and treatment of ESC and squmous epithelium precancerous lesion(SEPL) of esophagus is the major measure to decrease the incidence and improve the survival rate, and the key step of preventing ESC is early diagnosis and interdiction treatment of SEPL especially. Mucosal iodine staining is the key technique of early diagnosis of ESC and SEPL, but searching for its mechanism is lack, and lead to this technique could not be reformed and used widely. Many studies show: carcinogenesis in esophagus was a long and complex multistage process, esophageal squamous epithelium developed to tumors continuously and gradually, the course is from normal to hyperplasia to dysplasia and to cancer. Usually dysplasia of esophageal squamous epithelium is called precancerous lesion, which is the inevitable process of esophageal squamous cell carcinogensis. Mild dysplasia(mD) evolves slowly, has a low risk of developing cancer. The risk of developing cancer in moderate dysplasia(MD) is increase obviously, and the probability of severe dysplasia(SD) disappeared unaffectedly is few. So treatment of MD and SD is the key measure for controlling and preventing esophageal cancer, but now studies on chemoprevention of esophageal cancer by nutritional intervention or Chinese traditional medicine were only done in high risk people, and it's with high cost and low income, and effective treating method for MD and SD have not been found. According to above problems, we designed the study to research mechanism of esophageal mucosal iodine staining, so as to improve the detection rate of SEPL of esophagus. In order to research local treatment of SEPL of esophagus, we established the models of SEPL on rabbit's cheek mucosa and rhesus monkey's esophageal mucosa, discussed the interdictive and reverse treatment by local drugs in the models, and the scientific elements and clinic application of interdicting by endoscopic micro ravage method, and made a good progress in prevention of esophageal cancer. Methods 1. Study on mechanism of esophageal mucosal iodine staining. The esophageal mucosa of early esophageal cancer(EEC) and dysplasia cases and resected esophageal specimens of these cases were stained by combination iodine solution, tissues were taken from stained area, light stained area and unstained area of resected specimens respectively. All the taken tissues were dehydrated, fixed, marinated in olefin, embedded, sliced up, and these slices stained by HE, Langhan iodine and PAS, stained by Langhan iodine and PAS after digested with amylase. The results of esophageal mucosa iodine staining in vivo and vitro were compared with hisologcal slices stained by Langhan iodine and PAS. 2. Establishing a model of SEPL on rabbit's cheek mucousa and researching of its reverse treatment. Dimethylbenzanthracene (DMBA) only or combining with croton oil or nicotine were daubed or artifical membranes containing DMBA were pasted on rabbit's buccal mucosa directly for 12weeks. After an intermission of 4 weeks, tested rabbits were pasted membrane containing all-trans retinoic acid(ATRA) on buccal mucosa or were injected Kanglaite into submucosa for 12 weeks. Control groups were designed in both two steps. In the 16th and 28th week of the whole time, biopsies were performed at the tested area,histological examination was given by microscope, the ultrastructure was observed by electron microscope, and the proliferation index and apoptosis rate were tested by flow cytometry analysis, and the expression of P53,PCNA,Bcl-2 protein were exhibited by immunohistochemical staining. 3. Establishing a model of SEPL on rhesus monkey's esophagus DMBA was injected into 3 rhesus monkeys'esophageal submucosa by endoscope. 3 testing points and 2 control points were designed on each monkey's esophagus, there were 9 testing points and 6 control points totally. Testing points of each monkey were injected with 1%,0.5%,0.25% DMBA olefin liquid respectively, the dose for each time was 0.5ml and one time in 2 weeks,control points were injected with olefin liquid only in the same way. At the third and sixth month biopsies were performed at all injected points and histological examination were done。4. Experiment study of Argon plasma coagulation (APC) The experiment of APC on the pig's esophagus and the resected esophagus of the humen were divided into two groups. One group was injected with 0.9% sodium chloride or 50% glucose or distilled water before APC, another group was not injected before APC. The parameters of power setting were 40W,60W,99W, distance from top of probe to esophageal mucosa were 1or 2mm, the pulse durations were 1 or 3 secends, and the flow rate of argon were 1.6L/min. Pigs were killed on the third or seventh day after testing and the esophageal specimens were taken out. The mucosal color of coagulative area of specimens was observed, the depth and acreage of destroyed part of all specimens were measured, data of depths and acreage were analyzed statistically. 5. Clinical comparative study of treating esophageal precancerous lesion by APC Cases of MD and SD detected by endoscopy were divided in two groups. Cases of testing group were treated with APC,and control group treated with Zengshengping. After submocosal injection of 0.9% sodium chloride containing epinephrine, cases in testing group were treated with APC set 40Wof power setting, 1.6L/min of argon flow rate, and 1s~3s durations. If the lesion was destroyed incompletely, a retreatment would be needed one month later, then total treating times of each case were 1-5times. During operating, depending on the mucosa's color, depth of tissue damage area was judged. All cases were followed up by endoscopy in the 1th, 3th, 6th and 12th month and one time each year in subsequent time。6. Study on endoscopic mucosal resection (EMR) of esophageal precancerous lesion Cases with MD and SD of esophagus were diagnosed by endoscopy. If the lesion of them was less than 3cm it was resected using a cap by endoscope,and the cut specimens were examined histologically. If the edge of the specimens is normal tissue we believed lesions were removed completely. All cases were followed up by endoscopy in the 1th, 3th, 6th and 12th month and one time each year in subsequent time. Results 1. In tissue slice, surface layer and acanthos layer cell of nomal esophageal epithelium were purple after stained by PAS,and were brownish-yellow after stained by Langhan. The color became lighter and lighter from nomal esophageal muocsa to precancerous lesion and to cancer. The result of histochemical staining was consistent with those esophageal mucosal iodine staining in vivo and vitro. After tissues slice were digested by amylase, no color was appeared in the corresponding position, which suggested that glycogen in epithelium cells was the substantial basis for iodine staining. 2. Structure of rabbit's buccal mucosal squamous epithelium was similar with esophageal squamous epithelium of the humen. During cancer inducing, rabbit's buccal mucosa became roughness and thickness gradually, Leukodermas and few granule occurred on mucosa. In the 16th week , dysplasia were found on 47 rabbits (48 rabbits in testing group),and 64.5%(31/47)were MD and SD. In control group the dysplasia was found only on 1 of 12 rabbits, there were significant difference in two groups (p<0.05). Positive expression of P53,PCNA and Bcl-2 protein were found in rabbit'sbuccal mucosa tissues,and has a great correlation with the degree of dysplasia, especially in PCNA. Positive expression rate of PCNA and Bcl-2 were different between SD and hyperplasia (p<0.05). In flow cytometry analysis, proliferation index and apoptosis rate were increased with the lesions developing. In the 28th week,roughness and thickness of rabbit's buccal mucosa in reverse group became weaken, leukodermas were reduced and granules disappeared, and grades of dysplasia were reduced in histology. Seven of 16 rabbits treated with ATRA became better,the reversing result was better than control group(P<0.05), five of 16 rabbits treated with Kanglaite became better(P>0.05). The changes of epithelium cells ultrastructure be observed by TEM and SEM, and its chages in precancerous lesion were similar with the human. PCNA and Bcl-2 depressed evidently in both reverse group (P<0.05),P53 was increased in the group treated with Kanglaite (P<0.05). Proliferation index reduced and apoptosis rate increased in both reverse group (P<0.01). 3. Structure of rhesus monkey's esophageal squamous epithelium was more similar with the humen. Esophageal mucosa of testing points became roughness, thickness and turbidness. In the third month, mD appeared at 2 of 3 points given 1%DMBA, and at 1 of 3 points given 0.5%DMBA, the other testing points were hyperplasia, control points were normal or hyperplasia . 4. Coagulated breakage of pigs'esophageal mucousa and resected esophageal specimens of human were similar after APC. The form of damaged section looked like ellipse, size was from 5mm×4mm to 10mm×9mm, and its colors changed disciplinarily, the edge of damaged area was white and changed to brown-yellow gradually and became to black in center. Observed by microscope, edge of damaged area was epithelium, next part was mucosa and center part was submucosa. Comparing with mucosal color, in the white area only epithelium was destroyed, in brown-yellow area the mucosa was destroyed and in the black area the tissue damage extended to submucosa or muscle. So the changes of color were correlation with the depth of tissue damaged. By statistical analysis,depth of damaged tissue increases with theincreasing of power setting and pulse duration(P <0,001), decreases with the increasing of distance(P <0.05),and injection makes the damage much shallow(P <0.001), but there is no difference between three different drugs. 5. In testing group, 68 of 81 cases treated by APC were followed up for more than 6 months, In 56 cases of them the lesion disappeared, short-time cure rate was 82.35%, and 12 cases the lesion diminished and were treated again by APC. 57 cases were followed up for more than 12 months, in 52 cases of them the lesion disappeared and cure rate was 91.23%, 10 of 52 were retreatred cases, if they were subtracted, the cure rate was 73.68%. And during the whole following up period there were not cases the lesion kept fixedness or evolved or become cancer. In control group, 43 cases were followed up for more than one year, when followed up 6 months, lesions of 6 cases became better, of 28 kept fixedness,of 4 evolved,and of 5 developed to cancer, there was not case the lesion disappeared, and when followed up 12 months 8 cases became better, 21 kept fixedness, 6 evolved, and 7 developed to cancer in all. effects of all cases between two groups were marked different(P<0.0001). In testing group, light stricture happened in one case, but there were no bleeding and perforation. 6. EMR was performed with a cap in 36 patients. The lesions were resected completely in 32 patients(88.89%). The probability of removed completely decreased with the increasing of lesion size. After operated, histological results of 13 cases(36.11%) were not consistent with primary results, 7 SD cases (28.0%) were diagnosed as cancer after operation, 2 MD cases(18.18%) were diagnosed as SD and 1 (9.09%) was diagnosed as cancer. Endoscopic follow-up was carried out in 3 patients for more than 5 years, 9 patients for 3-5 years and 16 patients for 1-3 years, 8 patients for less than 1 year, no recurrence occurred during the period, 3 cases new lesions were appeared in other area, 1 patients died of other diseases, Oozing bleeding occurred in 1 cases after resection, but there was no perforation and stricture. Conclusions 1. Glycogen should be the substantial foundation for esophageal mucosaiodine staining, because combining of glycogen with iodine the color of the esophageal mucosa is changed. 2. Quantity of glycogen decreased from nomal esophageal squamous epithelium to dysplasia to cancer gradually, so the staining of esophageal mucosa die out. These are the basic elements for iodine staining technique in the clinical diagnosis of EEC and SEPL. 3. Pointing out an idea of reverse esophageal precancerous lesion by using drugs locally, and it was approved infeasible by animal testing. 4. Model of SEPL on rabbit's buccal mucosa was established successfully, this model is close to human being's esophageal SEPL, in this testing the technical operating is simply and the outcomes are observed easily. It set up a worktable for study of reversing SEPL. 5. Using retinoic acid locally to reverse SEPL on rabbit's buccal mucosa is effective. 6. Injecting Kanglaite locally can suppress cancer cells proliferation evidently, and can reverse precancerous lesion too. 7. Model of rhesus monkey's esophageal SEPL was established by endoscopic injecting DMBA initially, this will set up a greatest worktable for studying of human's esophageal SEPL reversing by using drugs locally, and make a better animal model for studying the mechanism of esophageal SEPL lesion happening and developing. 8. A theory that changes of mucosal color after APC is related with the tissus destroyed depth was put forward firstly, and proving this knowledge is very important for supervising clinic practice of APC,and guess this knowledge can be used in other kind coagulation by endoscopy。9. Submucousal injection was used in APC firstly,this method can prevent destroyed excessively, perforation, mucosal haematoma, submucosal emphysema and embolism of air,if lidocaine was added in injection, aceh will be avoided availably, so submucosal injection is very important improving for APC。10. Safe and effective parameters of APC to treat esophageal SEPL were...
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