程序化冻存兔膀胱粘膜异体移植治疗尿道狭窄实验研究
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摘要
程序化冻存兔膀胱粘膜异体移植替代尿道成形治疗尿道狭窄实验研究
第一部分
模拟TUR环境建立新西兰大白兔尿道狭窄模型
目的：模拟TUR环境，研究TUR手术后尿道狭窄发生原因，探索新西兰大白兔尿道狭窄模型建立方法。
方法：将20只雄性新西兰大白兔随机分成两组，每组10只，平均体重(2．0±0．1)kg。在戊巴比妥钠溶液静脉注射麻醉后，第一组单纯剥除1．0cm左右尿道粘膜，逐层缝合后留置F-8导尿管并固定。第二组在第一组基础上再用双极电凝灼烧尿道粘膜创面，所有操作均在10倍光镜下，运用显微器械完成。术后观察两组实验兔，在饲养至一个月、两个月时分别行顺行膀胱尿道造影。在两月时进行手术部位组织切片H．E．染色光镜检查。
结果：第一组实验兔在术后饲养时死亡2只，第二组死亡1只，余下完成实验。术后1周左右，留置导尿管自行脱落。观察发现第二组术后一月后明显较第一组焦躁。术后一个月顺行膀胱尿道造影两组均未见明显狭窄。而术后两个月时第二组尿道造影9只均出现明显狭窄，尿道管腔变细，粘膜粗糙不连续，而第一组仅2只出现狭窄症状。造模成功实验兔手术部位组织切片H．E．染色光镜检查结果提示大量纤维结缔组织沉积，缺乏尿道上皮生长。
结论：TUR手术时过度的电灼伤是引起术后尿道狭窄的重要原因，模拟TUR环境电灼损伤尿道可以稳定建立兔尿道狭窄模型，可以用于长段尿道狭窄治疗的研究。
第二部分
程序化冷冻保存对兔膀胱粘膜免疫原性影响的实验研究
目的：研究程序化冷冻保存对兔膀胱粘膜免疫原性及其在同种异体移植后引起排斥反应的影响，并探索其可能机制。
方法：取6只新西兰兔，分为3对，每对实验兔随机标记为A或B，分别取膀胱粘膜(分为冻存和未冻存两组)和脾脏，进行混合淋巴细胞实验，检验膀胱粘膜免疫原性的变化。将12只新西兰兔制成尿道狭窄模型，用未冻存和冻存的膀胱粘膜分别进行管状覆盖移植，另取6只新鲜兔设正常对照，移植2周后，取被移植兔的血液和脾脏用于淋巴细胞增殖实验，检测程序化冷冻保存对兔膀胱粘膜引起的免疫排斥反应的影响；同时取移植部位的尿道，进行CD3、CD4和CD8免疫组化染色，检测同种异体移植造成的淋巴细胞浸润情况。另取6只正常新西兰兔的膀胱粘膜，分为未冻存粘膜组和冻存粘膜组，提取mRNA，对RLA-Ⅰ、RLA-Ⅱ和RLA-Ⅲ基因进行定量PCR检测。
结果：混合淋巴细胞实验表明经程序化冷冻的粘膜细胞刺激异体淋巴细胞增殖反应的强度明显比未冷冻组弱，表现出极显著的差异；淋巴细胞增殖实验表明受体接受未经程序化冷冻的粘膜移植之后，其血液淋巴细胞和脾淋巴细胞增殖速度明显高于冻存粘膜移植组；免疫组化染色表明，冷冻移植组CD3、CD8阳性率及积累光密度明显小于未冷冻移植组；定量PCR实验表明，三类RLA基因的表达在冻存前后没有明显差异。
结论：程序化冷冻降低了兔膀胱粘膜的免疫原性，减轻了其在同种异体移植后引起的免疫排斥反应的强度。
第三部分
冷冻兔膀胱粘膜管状覆盖治疗尿道狭窄实验研究
目的：明确程序化冷冻保存技术能够较好保护组织细胞的结构，并通过观察程序化冷冻处理的膀胱粘膜移植片治疗尿道狭窄的手术效果，并与未经处理的对照组作比较，明确冷冻后异体兔膀胱粘膜是替代尿道成形术的良好材料。
方法：取6只新西兰兔膀胱粘膜，将其分成冷冻组及未冷冻组。(1)比较冷冻前后膀胱粘膜片光镜下组织学结构改变。(2)比较冷冻前后膀胱粘膜片电镜下组织学结构改变。将12只雄性新西兰大白兔，平均体重(2．1±0．2)kg，分为两组，每组6只，制成尿道狭窄模型。另取6只兔子经3％的戊巴比妥钠溶液静脉麻醉后，每只取下新鲜膀胱粘膜裁剪成等大两块10*8mm粘膜片，其中一块经程序化冷冻保存。随机从两组实验兔中各取一只组成一组，冷冻组应用程序化冷冻处理后的兔膀胱粘膜治疗，手术方式采取粘膜管状替代成型，对照组应用未经处理的粘膜片治疗。(3)比较手术后2周两组手术部位大体标本。(4)比较手术后2周两组手术部位HE染色后病理切片结果。(5)比较术后2月两组实验兔尿道造影结果。(6)比较术后2月手术部位组织HE染色后病理切片结果。
结果：(1)冷冻前后膀胱粘膜片光镜下组织学结构未见明显破坏，但粘膜表面有少许细胞脱落。(2)冷冻前后膀胱粘膜片电镜下组织学结构未见明显改变。(3)术后2周实验组手术部位炎症反应明显较对照组轻。(4)术后2周HE染色后病理切片结果提示冷冻组粘膜上皮细胞生长较好，而对照组上皮细胞脱落明显。(5)手术后2月行顺行膀胱尿道造影提示实验组尿道连续性以及管径直径明显优于对照组；(6)术后2月将手术部位切下后行HE染色，提示冷冻组尿道手术部位爬覆着一层鳞行上皮，而对照组因炎症反应剧烈，瘢痕组织较多见。
结论：应用程序化冷冻保存技术处理过的异体膀胱粘膜结构保存较好，用于治疗尿道狭窄手术的成功率较高，可以作为替代尿道成形术的修补材料，是对组织工程技术的很好的补充。
Experimental study on urethraplasty using allograft of programmedfrozen-thawed New Zealand rabbit's bladder mucosa
PARTⅠ
Establishment of a stable urethral stricture model by simulating the TURenvironment in New Zealand rabbits
Objective:To get the reason for iatrogenic urethral stricture after TUR operation,and simulate the TUR environment to explore the method of building a stable urethralstricture model in New Zealand white rabbits.
Methods:Twenty male New Zealand white rabbits were randomly divided into twogroups,each group had 10 rabbits with the average weight of (2.0±0.1)kg.Afterintravenous anesthesia by sodium pentobarbital,we made models by stripping 1.0 cmlong urethral mucosa in groupⅠ,then suturing the incision and fixing the F 8 catheter.In models of groupⅡ,we repeated coagulating the urethral mucosa wound by bipolarcoagulation.We did microsurgery for all models by 10 times Optical microscope.Weobserved the daily micturation condition after operation for both groups.We did theantegrade urethrography at one month and two months postoperatively.Then weobserved the histological change of surgical urethra.
Results:Two rabbits died after operation in groupⅠand one in groupⅡ,theremaining rabbits completed the test.One week later the catheter withdraweditself.We found the rabbits in groupⅡwere more irritable than those in groupⅠfromthe second month.At one month postoperatively,we did not find significance urethralstricture in both groups on antegrade urethrogram.But at two months postoperatively,we found significance urethral stricture with narrow lumen and discontinuous mucosa,for all 9 rabbits in groupⅡand only 2 in groupⅠ.We observed the histological changeof the surgical urethra,there was no evidence of epithelial cell growth but collagenfiber deposition.
Conclusion:Repeated coagulation is an important reason for iatrogenic urethral stricture after TUR.we establish a way to build a stable、homogeneous and repetitiveurethral stricture model of new Zealand rabbits by simulating the TUR environment,help to research and treat urethral stricture.
PARTⅡ
The effect of programmed cryopreservation on immunogenicity ofNew Zealand rabbit bladder mucosa
Objective:To evaluate the effect of programmed cryopreservation on immunogenicityof rabbit bladder mucosa and the intensity of immunological rejection caused by theallograft.
Methods:Six New Zealand white rabbits were divided into three pairs.Each one ofevery pair was randomly marked as A or B.The immunogenicity was examinedthrough mixed lymphocyte cell response using the bladder mucosa (cryopreservationand non-cryopreservation)and spleen from pair of rabbits.Twelve New Zealandrabbit models of urethral stricture were prepared to accept the transplant of bladdermucosa,which were divided into two groups of cryopreservation andnon-cryopreservation group,another six normal rabbits were made as controlgroup.Two weeks later,The lymphocyte proliferation was detected aftertransplantation from transplanted rabbit blood and spleen.At the same time,theurethra from transplanted rabbit for immunohistochemical staining was performed,and the expressions of CD3、CD4 and CD8 were observed.The mRNA of bladdermucosa (cryopreservation and non-cryopreservation)from another six New Zealandrabbits was extracted and the expressions of RLA-Ⅰ、RLA-Ⅱand RLA-Ⅲgenewere detected by real-time PCR.
Results:The ability of cryopreserved bladder epithelial cells stimulating allogeneiclymphocyte proliferation from the cryopreservation group was significantly weakerthan that from the non-cryopreservation group.The blood and spleen lymphocytesfrom transplanted rabbit bladder mucosa without cryopreservation showed higherproliferation rate than those with cryopreservation.Compared with thenon-cryopreservation group,the expression of CD3+ and CD8+ T cells infiltrated inthe transplanted locus of bladder mucosa was decreased in the cryopreservation group.The expressions of RLA genes didn't change significantly after cryopreservation.
Conclusion:Programmed cryopreservation of rabbit bladder mucosa could reduce itsimmunogenicity in the allotransplantation.and thus restrict the extent ofimmunological rejection.
PARTⅢ
Experimental study on the treatment of urethral stricture using tubularizedrabbit bladder mucosa after programmed cryopreservation
Objective:To confirm that cryopreservation technique is able to protect tissuestructure from the ultra low temperature environment.And to observe the surgicaleffect of substitution urethroplasty using allograft cryopreserved rabbit bladdermucosa,and compared with control group,in order to confirm allograft cryopreservedbladder mucosa is good material for substitution urethroplastyMethods:We harvested bladder mucosa from six New Zealand rabbits,then dividedinto two groups(cryopresevation and non-cryopresevation),(1)we compared thehistological change of H.E.stained bladder mucosa before and aftercryopreservation.(2)we compared the electron microscopic change of bladder mucosabefore and after cryopreservation.12 models were randomly divided into two groups,6 rabbits in each group with the average weight of (2.1±0.2)kg.Then we tookanother 6 rabbits,after intravenous anesthesia by 3% sodium pentobarbital,weharvested two pieces of fresh bladder mucosa (10*8mm),and one piece should beprogrammed cryopreserved.In test group,we performed the tubularized graftsubstitution urethroplasty using cryopreserved bladder mucosa for urethral stricture,and in control group we did the same operation but using non-cryopreserved bladdermucosa from the same rabbit.(3)we compared the urethral sample of surgical part atone week postoperatively.(4)we compared the histological changes of H.E.stainedurethral sample at one week postoperatively.(5)we compared the result of antegradeurethrography at two months postoperatively.(6)we compared the H.E.stainedurethral sample at two months postoperatively.
Results:There was no obvious histological change and electron microscopic changeof bladder mucosa before and after cryopreservation,but still a few epithelial cellswere shedding.The inflammation reaction of surgical part in test group was obviouslyharder than those in control group in one week after surgery.According to the resultsof antegrade urethrography,urethral continence and lumen diameter were better than those in control group in two months after surgery.Pathological result suggested thattwo weeks after surgery the urethral epithelial cells were growing well,but in controlgroup lots of epithelial cells were necrosis.In two months later there was a lay ofurethral epithelial in test group,but in control group we could only see scar because ofthe severe inflammation.
Conclusion:Programmed cryopreserved technique is able to maintain the mucosastructure.Because of the high success rate of surgery,we conclude that cryopreservedbladder mucosa could be used as an allograft substitution for urethroplasty in thetreatment of urethral stricture.
第一部分
模拟TUR环境建立新西兰大白兔尿道狭窄模型
目的：模拟TUR环境，研究TUR手术后尿道狭窄发生原因，探索新西兰大白兔尿道狭窄模型建立方法。
方法：将20只雄性新西兰大白兔随机分成两组，每组10只，平均体重(2．0±0．1)kg。在戊巴比妥钠溶液静脉注射麻醉后，第一组单纯剥除1．0cm左右尿道粘膜，逐层缝合后留置F-8导尿管并固定。第二组在第一组基础上再用双极电凝灼烧尿道粘膜创面，所有操作均在10倍光镜下，运用显微器械完成。术后观察两组实验兔，在饲养至一个月、两个月时分别行顺行膀胱尿道造影。在两月时进行手术部位组织切片H．E．染色光镜检查。
结果：第一组实验兔在术后饲养时死亡2只，第二组死亡1只，余下完成实验。术后1周左右，留置导尿管自行脱落。观察发现第二组术后一月后明显较第一组焦躁。术后一个月顺行膀胱尿道造影两组均未见明显狭窄。而术后两个月时第二组尿道造影9只均出现明显狭窄，尿道管腔变细，粘膜粗糙不连续，而第一组仅2只出现狭窄症状。造模成功实验兔手术部位组织切片H．E．染色光镜检查结果提示大量纤维结缔组织沉积，缺乏尿道上皮生长。
结论：TUR手术时过度的电灼伤是引起术后尿道狭窄的重要原因，模拟TUR环境电灼损伤尿道可以稳定建立兔尿道狭窄模型，可以用于长段尿道狭窄治疗的研究。
第二部分
程序化冷冻保存对兔膀胱粘膜免疫原性影响的实验研究
目的：研究程序化冷冻保存对兔膀胱粘膜免疫原性及其在同种异体移植后引起排斥反应的影响，并探索其可能机制。
方法：取6只新西兰兔，分为3对，每对实验兔随机标记为A或B，分别取膀胱粘膜(分为冻存和未冻存两组)和脾脏，进行混合淋巴细胞实验，检验膀胱粘膜免疫原性的变化。将12只新西兰兔制成尿道狭窄模型，用未冻存和冻存的膀胱粘膜分别进行管状覆盖移植，另取6只新鲜兔设正常对照，移植2周后，取被移植兔的血液和脾脏用于淋巴细胞增殖实验，检测程序化冷冻保存对兔膀胱粘膜引起的免疫排斥反应的影响；同时取移植部位的尿道，进行CD3、CD4和CD8免疫组化染色，检测同种异体移植造成的淋巴细胞浸润情况。另取6只正常新西兰兔的膀胱粘膜，分为未冻存粘膜组和冻存粘膜组，提取mRNA，对RLA-Ⅰ、RLA-Ⅱ和RLA-Ⅲ基因进行定量PCR检测。
结果：混合淋巴细胞实验表明经程序化冷冻的粘膜细胞刺激异体淋巴细胞增殖反应的强度明显比未冷冻组弱，表现出极显著的差异；淋巴细胞增殖实验表明受体接受未经程序化冷冻的粘膜移植之后，其血液淋巴细胞和脾淋巴细胞增殖速度明显高于冻存粘膜移植组；免疫组化染色表明，冷冻移植组CD3、CD8阳性率及积累光密度明显小于未冷冻移植组；定量PCR实验表明，三类RLA基因的表达在冻存前后没有明显差异。
结论：程序化冷冻降低了兔膀胱粘膜的免疫原性，减轻了其在同种异体移植后引起的免疫排斥反应的强度。
第三部分
冷冻兔膀胱粘膜管状覆盖治疗尿道狭窄实验研究
目的：明确程序化冷冻保存技术能够较好保护组织细胞的结构，并通过观察程序化冷冻处理的膀胱粘膜移植片治疗尿道狭窄的手术效果，并与未经处理的对照组作比较，明确冷冻后异体兔膀胱粘膜是替代尿道成形术的良好材料。
方法：取6只新西兰兔膀胱粘膜，将其分成冷冻组及未冷冻组。(1)比较冷冻前后膀胱粘膜片光镜下组织学结构改变。(2)比较冷冻前后膀胱粘膜片电镜下组织学结构改变。将12只雄性新西兰大白兔，平均体重(2．1±0．2)kg，分为两组，每组6只，制成尿道狭窄模型。另取6只兔子经3％的戊巴比妥钠溶液静脉麻醉后，每只取下新鲜膀胱粘膜裁剪成等大两块10*8mm粘膜片，其中一块经程序化冷冻保存。随机从两组实验兔中各取一只组成一组，冷冻组应用程序化冷冻处理后的兔膀胱粘膜治疗，手术方式采取粘膜管状替代成型，对照组应用未经处理的粘膜片治疗。(3)比较手术后2周两组手术部位大体标本。(4)比较手术后2周两组手术部位HE染色后病理切片结果。(5)比较术后2月两组实验兔尿道造影结果。(6)比较术后2月手术部位组织HE染色后病理切片结果。
结果：(1)冷冻前后膀胱粘膜片光镜下组织学结构未见明显破坏，但粘膜表面有少许细胞脱落。(2)冷冻前后膀胱粘膜片电镜下组织学结构未见明显改变。(3)术后2周实验组手术部位炎症反应明显较对照组轻。(4)术后2周HE染色后病理切片结果提示冷冻组粘膜上皮细胞生长较好，而对照组上皮细胞脱落明显。(5)手术后2月行顺行膀胱尿道造影提示实验组尿道连续性以及管径直径明显优于对照组；(6)术后2月将手术部位切下后行HE染色，提示冷冻组尿道手术部位爬覆着一层鳞行上皮，而对照组因炎症反应剧烈，瘢痕组织较多见。
结论：应用程序化冷冻保存技术处理过的异体膀胱粘膜结构保存较好，用于治疗尿道狭窄手术的成功率较高，可以作为替代尿道成形术的修补材料，是对组织工程技术的很好的补充。
Experimental study on urethraplasty using allograft of programmedfrozen-thawed New Zealand rabbit's bladder mucosa
PARTⅠ
Establishment of a stable urethral stricture model by simulating the TURenvironment in New Zealand rabbits
Objective:To get the reason for iatrogenic urethral stricture after TUR operation,and simulate the TUR environment to explore the method of building a stable urethralstricture model in New Zealand white rabbits.
Methods:Twenty male New Zealand white rabbits were randomly divided into twogroups,each group had 10 rabbits with the average weight of (2.0±0.1)kg.Afterintravenous anesthesia by sodium pentobarbital,we made models by stripping 1.0 cmlong urethral mucosa in groupⅠ,then suturing the incision and fixing the F 8 catheter.In models of groupⅡ,we repeated coagulating the urethral mucosa wound by bipolarcoagulation.We did microsurgery for all models by 10 times Optical microscope.Weobserved the daily micturation condition after operation for both groups.We did theantegrade urethrography at one month and two months postoperatively.Then weobserved the histological change of surgical urethra.
Results:Two rabbits died after operation in groupⅠand one in groupⅡ,theremaining rabbits completed the test.One week later the catheter withdraweditself.We found the rabbits in groupⅡwere more irritable than those in groupⅠfromthe second month.At one month postoperatively,we did not find significance urethralstricture in both groups on antegrade urethrogram.But at two months postoperatively,we found significance urethral stricture with narrow lumen and discontinuous mucosa,for all 9 rabbits in groupⅡand only 2 in groupⅠ.We observed the histological changeof the surgical urethra,there was no evidence of epithelial cell growth but collagenfiber deposition.
Conclusion:Repeated coagulation is an important reason for iatrogenic urethral stricture after TUR.we establish a way to build a stable、homogeneous and repetitiveurethral stricture model of new Zealand rabbits by simulating the TUR environment,help to research and treat urethral stricture.
PARTⅡ
The effect of programmed cryopreservation on immunogenicity ofNew Zealand rabbit bladder mucosa
Objective:To evaluate the effect of programmed cryopreservation on immunogenicityof rabbit bladder mucosa and the intensity of immunological rejection caused by theallograft.
Methods:Six New Zealand white rabbits were divided into three pairs.Each one ofevery pair was randomly marked as A or B.The immunogenicity was examinedthrough mixed lymphocyte cell response using the bladder mucosa (cryopreservationand non-cryopreservation)and spleen from pair of rabbits.Twelve New Zealandrabbit models of urethral stricture were prepared to accept the transplant of bladdermucosa,which were divided into two groups of cryopreservation andnon-cryopreservation group,another six normal rabbits were made as controlgroup.Two weeks later,The lymphocyte proliferation was detected aftertransplantation from transplanted rabbit blood and spleen.At the same time,theurethra from transplanted rabbit for immunohistochemical staining was performed,and the expressions of CD3、CD4 and CD8 were observed.The mRNA of bladdermucosa (cryopreservation and non-cryopreservation)from another six New Zealandrabbits was extracted and the expressions of RLA-Ⅰ、RLA-Ⅱand RLA-Ⅲgenewere detected by real-time PCR.
Results:The ability of cryopreserved bladder epithelial cells stimulating allogeneiclymphocyte proliferation from the cryopreservation group was significantly weakerthan that from the non-cryopreservation group.The blood and spleen lymphocytesfrom transplanted rabbit bladder mucosa without cryopreservation showed higherproliferation rate than those with cryopreservation.Compared with thenon-cryopreservation group,the expression of CD3+ and CD8+ T cells infiltrated inthe transplanted locus of bladder mucosa was decreased in the cryopreservation group.The expressions of RLA genes didn't change significantly after cryopreservation.
Conclusion:Programmed cryopreservation of rabbit bladder mucosa could reduce itsimmunogenicity in the allotransplantation.and thus restrict the extent ofimmunological rejection.
PARTⅢ
Experimental study on the treatment of urethral stricture using tubularizedrabbit bladder mucosa after programmed cryopreservation
Objective:To confirm that cryopreservation technique is able to protect tissuestructure from the ultra low temperature environment.And to observe the surgicaleffect of substitution urethroplasty using allograft cryopreserved rabbit bladdermucosa,and compared with control group,in order to confirm allograft cryopreservedbladder mucosa is good material for substitution urethroplastyMethods:We harvested bladder mucosa from six New Zealand rabbits,then dividedinto two groups(cryopresevation and non-cryopresevation),(1)we compared thehistological change of H.E.stained bladder mucosa before and aftercryopreservation.(2)we compared the electron microscopic change of bladder mucosabefore and after cryopreservation.12 models were randomly divided into two groups,6 rabbits in each group with the average weight of (2.1±0.2)kg.Then we tookanother 6 rabbits,after intravenous anesthesia by 3% sodium pentobarbital,weharvested two pieces of fresh bladder mucosa (10*8mm),and one piece should beprogrammed cryopreserved.In test group,we performed the tubularized graftsubstitution urethroplasty using cryopreserved bladder mucosa for urethral stricture,and in control group we did the same operation but using non-cryopreserved bladdermucosa from the same rabbit.(3)we compared the urethral sample of surgical part atone week postoperatively.(4)we compared the histological changes of H.E.stainedurethral sample at one week postoperatively.(5)we compared the result of antegradeurethrography at two months postoperatively.(6)we compared the H.E.stainedurethral sample at two months postoperatively.
Results:There was no obvious histological change and electron microscopic changeof bladder mucosa before and after cryopreservation,but still a few epithelial cellswere shedding.The inflammation reaction of surgical part in test group was obviouslyharder than those in control group in one week after surgery.According to the resultsof antegrade urethrography,urethral continence and lumen diameter were better than those in control group in two months after surgery.Pathological result suggested thattwo weeks after surgery the urethral epithelial cells were growing well,but in controlgroup lots of epithelial cells were necrosis.In two months later there was a lay ofurethral epithelial in test group,but in control group we could only see scar because ofthe severe inflammation.
Conclusion:Programmed cryopreserved technique is able to maintain the mucosastructure.Because of the high success rate of surgery,we conclude that cryopreservedbladder mucosa could be used as an allograft substitution for urethroplasty in thetreatment of urethral stricture.
引文
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