受体上皮细胞灌注后大鼠气管异位移植再上皮化的实验研究
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摘要
背景
现有的技术条件下,气管环形切除的长度一般不宜超过成人气管的1/2(约6cm)、婴幼儿气管的1/3,否则将无法实施端端吻合。而更长段气管的切除后重建则需通过同种异体气管移植、人工或组织工程气管植入或自体组织再造来完成。相比较而言,同种异体气管移植具有相当的优势,因此始终是气管外科领域中的热点。但是,同种异体气管移植目前仍主要处于实验研究阶段,成功的临床病例报道非常少,均为单一病例报道,而且往往缺乏长期的随访结果。这其中主要的困难之一就是免疫排斥。而免疫抑制治疗虽然能够提高移植成功率,但同时也大大增加了感染和罹患肿瘤的可能性,因此不少学者都在寻找新途径来抑制移植免疫排斥反应,而其中方法之一便是直接降低供体气管的抗原性。
研究发现,去除供体气管的上皮细胞能够消除或者减弱气管组织的抗原性,从而抑制移植免疫排斥反应,而无需或仅需低剂量的免疫抑制治疗。但是,气管上皮细胞不但对于恢复气管正常功能具有重要意义,而且在移植过程中也发挥着关键作用。因此,理想的解决方法是：以受体上皮细胞取代供体气管原有的上皮细胞。也就是说,在去除供体气管的上皮细胞之后,采取措施促进受体源性的再上皮化。从而既达到了降低气管组织抗原性的目的,又避免了上皮细胞丢失所造成的不良后果。
迄今为止,国外对于气管移植过程中供体裸气管内实现受体源性再上皮化的相关研究极少,而国内更是未见有类似的研究。因此,其中很多关键环节尚未得到清晰阐释,理论基础相当薄弱,技术方法也并不完备。本研究计划在胰酶冷消化法去除供体气管上皮细胞的同时,将受体气管上皮细胞进行体外培养,制备成细胞悬液后灌注于供体裸气管内,再异位移植于免疫功能正常的受体大鼠皮下,完成受体源性的再上皮化；从而对这一方法的实际效果进行客观的评价,并进一步优化现有的技术手段,同时详细勾勒出具体的演进过程,完善气管移植再上皮化的理论体系,为今后的大动物实验乃至临床研究奠定基础。
第一部分大鼠气管上皮细胞培养和灌注、供体裸气管制备和气管异位移植模型建立
目的
本研究设想在消除供体气管上皮组织之后,将受体气管上皮细胞种植在供体裸气管内再进行移植,从而在降低供体气管免疫原性的同时完成受体源性的再上皮化,达到气管成功移植的目的。而其中的关键点在于供体裸气管的制备、气管上皮细胞的体外培养和气管内灌注、相关动物模型的建立。
方法
上皮细胞培养及细胞悬液制备：采用无血清完全F12培养基对SD大鼠气管上皮细胞进行体外培养,并调整细胞浓度,使其达到10×106/ml。
供体大鼠裸气管制备：以Wistar大鼠(n=80)作为供体,取长约1.5-2.0cm的气管,一端结扎后灌入0.25%的胰酶溶液至气管微涨,再用丝线将另一端结扎,放入4℃冰箱18小时后取出,进行组织学切片HE染色检测上皮细胞消化结果,合格的裸气管以细胞培养液保存备用。
上皮细胞灌注及大鼠气管异位移植模型建立：结扎供体裸气管一端,灌入已制备好的细胞悬液,再结扎另一端。以SD大鼠(n=80)作为受体,在背部皮肤剪一小口后,于皮下游离一隧道,隧道顶端位于项部。将灌有上皮细胞悬液的供体气管置入隧道顶端皮下,缝合皮肤切口后进行饲养。
结果
上皮细胞培养及细胞悬液制备：气管上皮细胞为圆形,折光度好,悬浮在培养液中。24小时后可见细胞大部分开始贴壁；3天后见细胞贴壁呈现铺路石样；4-5天后细胞增殖成片状的细胞团。
供体裸气管制备：HE染色提示,供体气管上皮细胞完全被去除,基底膜完整,软骨组织正常。
上皮细胞灌注及大鼠气管异位移植模型建立：每例模型建立平均用时5.2±1.5分钟。全组(n=80)受体动物术后无一例死亡,生存率为100%,亦未出现感染等术后并发症。
结论
采用胰酶冷消化法能够安全、彻底去除气管上皮细胞,而对其它组织基本无损伤；改进型无血清完全型F12培养方法体外培养上皮细胞培养效果良好,是一种相对简单、实用、有效的培养方法；细胞悬液灌注进行上皮细胞在气管内的种植,操作十分简便,能够很好的保留细胞悬液,为上皮细胞的贴壁、生长和分化创造了条件；大鼠背部皮下气管异位移植模型操作简单、存活率高。
第二部分免疫抑制治疗对受体上皮细胞灌注后大鼠气管异位移植再上皮化的影响
目的
上皮细胞被完全去除之后,气管其它组织依然存活,其中某些细胞和组织也存在有MHC抗原。这也就意味着供体裸气管仍然具有一定的免疫原性,移植后仍然有可能诱发一定程度的免疫排斥反应。而免疫排斥反应是否会对受体源性的再上皮化过程产生不利影响?是否需要实施免疫抑制治疗?免疫抑制治疗的最佳方案是什么?在适当条件下受体上皮细胞灌注后大鼠气管异位移植的最终结果如何?这些问题都需要通过进一步的实验来给予解答。
方法
大鼠气管异位移植模型建立后,将实验动物完全随机分为六组。对照组1(n=10)：供体裸气管内灌入生理盐水,不使用任何免疫抑制剂；对照组2(n=10)：供体裸气管内灌注受体上皮细胞悬液,不使用任何免疫抑制剂；实验组1(n=10)：供体裸气管内灌注受体上皮细胞悬液,移植后受体鼠每日肌肉注射FK506 (0.5 mg/kg/d),连续使用3天；实验组2(n=10)：供体裸气管内灌注受体上皮细胞悬液,移植后受体鼠每日肌肉注射FK506 (0.5 mg/kg/d),连续使用10天；实验组3(n=10)：供体裸气管内灌注受体上皮细胞悬液,移植后受体鼠每日肌肉注射FK506 (1.0 mg/kg/d),连续使用3天；实验组4(n=10)：供体裸气管内灌注受体上皮细胞悬液,移植后受体鼠每日肌肉注射FK506 (1.0 mg/kg/d),连续使用10天。移植后第28天,处死大鼠,取出移植气管,标本分切后分别进行以下检测：组织形态学评价、透射电镜观察、淋巴细胞浸润计数、上皮细胞分化鉴定(CK14、CK18和CFTR)、上皮细胞纤毛比例和气管壁纤维组织含量测定。
结果
实验组的总体情况明显优于对照组。其中,对照组1的结果最不理想,未见任何上皮细胞再生,而且管腔出现严重狭窄、闭塞；对照组2也并未实现再上皮化,但稍好于对照组1；实验组1和实验组2相比,各项检测结果基本相似：气管内壁仅在局部有上皮细胞再生,且细胞尚未分化成熟,从而提示在这两组中存在有受体源性的再上皮化,但结果并不理想；实验组3和实验组4的结果非常相近,同时也是最为理想的：受体源性再上皮化取得成功,气管内覆盖了完整连续的成熟的纤毛柱状上皮细胞,并具有腺体分泌功能、屏障防御功能。
结论
供体裸气管内灌注受体上皮细胞后再实施异位移植,在一定的条件下,能够成功实现受体源性的完全再上皮化；而在这一过程中,仍需进行短期、低剂量的免疫抑制治疗；使用FK506的最佳方案是在移植后连续使用3天,剂量为1.0mg/kg/d;上皮细胞,即便是尚未附壁的上皮细胞,可能对于移植后的阻塞性病变有抑制作用。
第三部分受体上皮细胞灌注后大鼠气管异位移植再上皮化过程的研究
目的
第二部分的实验已经证实,在短期、低剂量的应用免疫抑制剂的情况下,供体裸气管内灌注受体上皮细胞后再实施异位移植,能够成功实现受体源性的完全再上皮化。但是对于其具体的演进过程尚缺乏足够的了解。
方法
大鼠气管异位移植模型建立后,将实验动物完全随机分为六组,移植后所有受体鼠每日肌肉注射FK506 (1.0 mg/kg/d),连续使用3天。在移植后第3天、第7天、第14天、第21天、第28天、第35天,分别随机处死一组大鼠(n=10),取出移植气管,分切后进行以下检测：组织形态学评价、透射电镜观察、上皮细胞分化鉴定(CK14、CK18和CFTR)、上皮细胞增殖能力检测(Ki67)和上皮细胞纤毛比例测定。
结果
在移植后的第3天,气管内壁局部覆盖有无纤毛的扁平上皮细胞；透射电镜发现此处细胞结构正常,偶见有异常短小、稀疏的类纤毛样结构,细胞间隙宽大,未见明显的细胞连接；CK14、CK18和CFTR的表达均处于低水平,Ki67处于高水平,纤毛比例低。
第7天,气管内壁完整覆盖有无纤毛的扁平上皮细胞。其余结果与第3天相似。
第14天,上皮细胞层为扁平细胞和柱状细胞交替,透射电镜发现部分柱状细胞处呈现假复层结构,存在杯状细胞,柱状细胞表面可见少量纤毛,细胞间隙较窄,可见细胞连接；CK14和CFTR的表达仍处于低水平,CK18开始升高,Ki67开始下降,纤毛比例开始升高。
第21天,气管内壁覆盖完整的柱状上皮细胞,偶见刷状缘,透射电镜发现大部分细胞层为假复层结构,其余表现与第14天相似；CK14的表达仍处于低水平,CK18达到峰值,CFTR开始升高,Ki67继续下降,纤毛比例继续升高。
第28天,气管内壁覆盖完整的柱状上皮细胞,可见较明显的刷状缘,透射电镜发现假复层结构明显,可见杯状细胞、腺体分泌细胞、基底细胞等多种细胞类型,细胞间隙狭窄,细胞连接紧密；CK14和Ki67的表达处于低水平,CK18维持高水平,CFTR达到峰值,纤毛比例达到峰值。
第35天的表现与第28天相仿。
结论
受体上皮细胞灌注后大鼠气管异位移植再上皮化的演进过程为：灌注后细胞迅速附壁,其中成功附壁的上皮细胞开始增殖,进而完整覆盖气管内壁,形成单层扁平上皮组织,而后上皮细胞开始分化,最终成为成熟的假复层纤毛柱状上皮组织。
Background
In the end-to-end anastomosis of trachea, the resectable length is restricted to 1/2 of the total length (6 cm) in adults or 1/3 in children, and replacement of longer sections will only be feasible if a safe and functional tracheal replacement can be developed, such as tracheal allografts, artificial or tissue-engineered airway, autogenous tissues graft and so on. Among them, tracheal allografts may be the most effective option for achieving airway reconstruction at present. Although more and more researchers and clinicians focus on tracheal transplantations, there are a few clinical reports of tracheal allotransplantations. And immune rejection is one of key obstacles in tracheal allografts. Immunosuppressive therapy increases the possibility of sever infection and malignant tumor, while improving the allografts. Therefore it is necessary to find an alternative to suppress the immune rejection after tracheal transplantation.
As is known, the immune rejection after allografts can be suppressed by eliminating the antigenicity of grafts. It was reported that tracheal antigenicity may be eliminated or reduced by the removal of epithelium in trachea. However, epithelial tissues play a key role in functional reconstruction and transplantation of trachea. Therefore, it is the best way to promote reepithelization from the recipient after the removal of donor epithelium.
Heretofore, it is rare that the study of reepithelization from the recipient after the removal of donor epithelium in tracheal allografts. Thus the key steps in reepithelization process are not clear, the technique means are imperfect, and the theoretical basis is very poor. We plan to use enzymatic dissociation with trypsin to remove the epithelium from the rat donor trachea which is then colonized by epithelial cells that have been cultured from cells taken from the recipient. Afterwards, the donor trachea is heterotopically transplanted to the dorsum of recipient rat and reepithelization will be completed. And our objectives are to objectively evaluate the present method, to detailedly describe the whole process and to optimize the technique means.
PART ONE
Culture and perfusion of epithelial cells from rat trachea, production of donor nude trachea and establishment of heterotopic tracheal transplantation model
Objective
We plan to perfuse the epithelial cells cultured from the recipient trachea into the donor nude trachea in which donor epithelial tissues has been removed by enzymatic dissociation in order to reduce the antigenicity of donor trachea and complete reepithelization from the recipient. It is the key steps that culture and perfusion of epithelial cells from rat trachea, production of donor nude trachea and establishment of heterotopic tracheal transplantation model.
Methods
The tracheal epithelial cells of SD rats were primarily cultured in Ham's F-12 and the cultured cells were diluted to cell suspension (10×106 cells/ml).
Wistar rats (n=80) were taken as donors and SD rats (n=80) as recipients. Tracheas (1.5-2.0 cm) were excised from below the larynx to the major bronchi. The explanted tracheas were digested for 18 h with 0.25% trypsin at 4℃, then the qualification of donor nude tracheas were evaluated by HE stain. The qualified donor nude tracheas were stored in Ham's F-12. One end of donor nude trachea was ligated, and the recipient cell suspension was perfused into its lumen before ligation of another end. Afterwords, donor tracheas were heterotopically subcutaneous transplanted to the dorsum of recipients.
Results
Epithelial cells being round-shape have good diopter and suspended in culture solution. After 24 hours, the majority of cells started to adhere to the wall. After 3 days, cells showed typical "flagstone" appearance. After 4-5 days, cells formed the flake-shape cell clusters. HE stain showed that epithelial cells were totally removed from the donor trachea with integrated basement membrane and normal cartilage tissues. The mean time of animal model establishment is 5.2±1.5 min. There was no postoperative complications, eg. infection, and the survival rate was 100%.
Conclusion
The enzymatic dissociation with trypsin is a safe and effective method to remove epithelial cells from the donor trachea. Ham's F-12 is a suitable culture solution for our study. Perfusion of cells suspension is a simple and available technique to complete the epithelial implantation. Because of easy operation and high success rate, heterotopic subcutaneous tracheal transplantation model in rat is the best choice in present study.
PART TWO
Effect of immunosuppressive therapy on reepithelization after rat heterotopic tracheal graft with recipient epithelial cells perfusion
Objective
After totally removal of epithelial cells, there are still MHC antigens existing in rest cells and tissues, which means donor nude trachea still has antigenicity and immune rejection will take place after transplantation. And we need to answer the following questions:Will immune rejection has adverse influence on reepithelization from the recipient? Is it necessary to perform the immunosuppressive therapy? What is the best treatment regimen of immunosuppressive therapy? How is reepithelization after rat heterotopic tracheal graft with recipient epithelial cells prefusion?
Methods
After establishment of heterotopic subcutaneous tracheal transplantation model in rat, animals were randomly divided into six groups. Control group 1 (n=10):normal saline was perfused into donor nude trachea, without immunosuppressive therapy; Control group 2 (n=10):recipient epithelial cells suspension was perfused into donor nude trachea, without immunosuppressive therapy; Test group 1 (n=10):recipient epithelial cells suspension was perfused into donor nude trachea, with intramuscular injection of Tacrolimus (0.5 mg/kg/d) only for three consecutive days after transplantation; Test group 2 (n=10):recipient epithelial cells suspension was perfused into donor nude trachea, with intramuscular injection of Tacrolimus (0.5 mg/kg/d) for seven consecutive days after transplantation; Test group 3 (n=10):recipient epithelial cells suspension was perfused into donor nude trachea, with intramuscular injection of Tacrolimus (1.0 mg/kg/d) only for three consecutive days after transplantation; Test group 4 (n=10):recipient epithelial cells suspension was perfused into donor nude trachea, with intramuscular injection of Tacrolimus (1.0 mg/kg/d) for seven consecutive days after transplantation. All rats were killed on 28 days after transplantation and then the implanted tracheas were harvested. In all specimens, histological and ultrastructural changes were evaluated, infiltrated lymphocytes were counted, differentiation of epithelium (CK14, CK18, CFTR) were identified, percentage of cilia and fibrosis tissues were calculated.
Results
Generally the results of test groups were better than control groups. In the latter, there was not any epithelial regeneration, and tracheal stenosis, even lumen occlusion, were found. Test group 1 and test group 2 had the similar results that immature epithelial regeneration took place in the regional area of tracheal inner surface. And test group 3 and test group 4 also had the similar results that mature ciliated columnar epithelium covered the whole inner surface of trachea.
Conclusion
It is an effective way to complete reepithelization from the recipient after rat heterotopic tracheal graft with recipient epithelial cells injection. And short-term and low dosage immunosuppressive therapy is necessary. The best regimen is the intramuscular injection of Tacrolimus (1.0 mg/kg/d) for three consecutive days after transplantation. Moreover, epithelial cells from the recipient may inhabit the development of obliterated airway disease.
PART THREE
Study on the process of reepithelization after rat heterotopic tracheal graft with recipient epithelial cells perfusion
Objective
In part two, we have improved that it is an effective way to complete reepithelization from the recipient that heterotopic tracheal graft with recipient epithelial cells perfusion in rat with short-term and low dosage immunosuppressive therapy. But it is still unclear about the detailed process of reepithelization.
Methods
After establishment of heterotopic subcutaneous tracheal transplantation model in rat, the animals were randomly divided into six groups. All animals were intramuscular injected of Tacrolimus (1.0 mg/kg/d) only for three consecutive days after transplantation. Rats (n=10) were killed on 3,7,14,21,28 and 35 days respectively after transplantation and then the implanted tracheae were harvested. In all specimens, histological and ultrastructural changes were evaluated, differentiation (CK14, CK18, CFTR) and proliferation (Ki67) of epithelium were identified, percentage of cilia were calculated.
Results
On 3 days after transplantation, the regional areas of tracheal inner surface were covered by non-ciliated flate cells with normal cellular ultrastructure and wide intercellular space, but without cell junctions. And the levels of CK14, CK18, CFTR and percentage of cilia were low, while Ki67 was high.
On 7 days, the whole areas of tracheal inner surface were covered by non-ciliated flate cells. And the rest results were similar to those on 3 days after transplantation.
On 14 days, the whole areas of tracheal inner surface were covered by the mixture of flate cells and columnar cells with sparse cilia. The latter arranged in pseudostratified structure which consisted of goblet cells, basal cells and so on. Cell junctions were found under Transmission Electron Microscope. The levels of CK14, CFTR were still low, but the level of CK18 and percentage of cilia started to increase and the level of Ki67 started to decrease.
On 21 days, the whole areas of tracheal inner surface were covered by columnar cells. The majority of cells arranged in pseudostratified structure. The level of CK14 was still low, and percentage of cilia still increased and the level of Ki67 decreased. But the level of CFTR started to increase and CK18 reached the peak level.
On 28 days, the whole areas of tracheal inner surface were covered by ciliated columnar cells arranged in pseudostratified structure. The narrow intercellular space and tight cell junctions were observed under Transmission Electron Microscope. The levels of CK14, Ki67 were low, and the levels of CK18, CFTR and percentage of cilia were high.
The results on 35 days after transplantation were similar to those on 28 days.
Conclusion
Cells adhered to tracheal inner wall quickly after perfusion of cells suspension. Then the adherent flate cells started to proliferate until the whole inner surface was covered. And the cells started to differentiate. Finally, mature pseudostratified ciliated columnar cells appeared.
现有的技术条件下,气管环形切除的长度一般不宜超过成人气管的1/2(约6cm)、婴幼儿气管的1/3,否则将无法实施端端吻合。而更长段气管的切除后重建则需通过同种异体气管移植、人工或组织工程气管植入或自体组织再造来完成。相比较而言,同种异体气管移植具有相当的优势,因此始终是气管外科领域中的热点。但是,同种异体气管移植目前仍主要处于实验研究阶段,成功的临床病例报道非常少,均为单一病例报道,而且往往缺乏长期的随访结果。这其中主要的困难之一就是免疫排斥。而免疫抑制治疗虽然能够提高移植成功率,但同时也大大增加了感染和罹患肿瘤的可能性,因此不少学者都在寻找新途径来抑制移植免疫排斥反应,而其中方法之一便是直接降低供体气管的抗原性。
研究发现,去除供体气管的上皮细胞能够消除或者减弱气管组织的抗原性,从而抑制移植免疫排斥反应,而无需或仅需低剂量的免疫抑制治疗。但是,气管上皮细胞不但对于恢复气管正常功能具有重要意义,而且在移植过程中也发挥着关键作用。因此,理想的解决方法是：以受体上皮细胞取代供体气管原有的上皮细胞。也就是说,在去除供体气管的上皮细胞之后,采取措施促进受体源性的再上皮化。从而既达到了降低气管组织抗原性的目的,又避免了上皮细胞丢失所造成的不良后果。
迄今为止,国外对于气管移植过程中供体裸气管内实现受体源性再上皮化的相关研究极少,而国内更是未见有类似的研究。因此,其中很多关键环节尚未得到清晰阐释,理论基础相当薄弱,技术方法也并不完备。本研究计划在胰酶冷消化法去除供体气管上皮细胞的同时,将受体气管上皮细胞进行体外培养,制备成细胞悬液后灌注于供体裸气管内,再异位移植于免疫功能正常的受体大鼠皮下,完成受体源性的再上皮化；从而对这一方法的实际效果进行客观的评价,并进一步优化现有的技术手段,同时详细勾勒出具体的演进过程,完善气管移植再上皮化的理论体系,为今后的大动物实验乃至临床研究奠定基础。
第一部分大鼠气管上皮细胞培养和灌注、供体裸气管制备和气管异位移植模型建立
目的
本研究设想在消除供体气管上皮组织之后,将受体气管上皮细胞种植在供体裸气管内再进行移植,从而在降低供体气管免疫原性的同时完成受体源性的再上皮化,达到气管成功移植的目的。而其中的关键点在于供体裸气管的制备、气管上皮细胞的体外培养和气管内灌注、相关动物模型的建立。
方法
上皮细胞培养及细胞悬液制备：采用无血清完全F12培养基对SD大鼠气管上皮细胞进行体外培养,并调整细胞浓度,使其达到10×106/ml。
供体大鼠裸气管制备：以Wistar大鼠(n=80)作为供体,取长约1.5-2.0cm的气管,一端结扎后灌入0.25%的胰酶溶液至气管微涨,再用丝线将另一端结扎,放入4℃冰箱18小时后取出,进行组织学切片HE染色检测上皮细胞消化结果,合格的裸气管以细胞培养液保存备用。
上皮细胞灌注及大鼠气管异位移植模型建立：结扎供体裸气管一端,灌入已制备好的细胞悬液,再结扎另一端。以SD大鼠(n=80)作为受体,在背部皮肤剪一小口后,于皮下游离一隧道,隧道顶端位于项部。将灌有上皮细胞悬液的供体气管置入隧道顶端皮下,缝合皮肤切口后进行饲养。
结果
上皮细胞培养及细胞悬液制备：气管上皮细胞为圆形,折光度好,悬浮在培养液中。24小时后可见细胞大部分开始贴壁；3天后见细胞贴壁呈现铺路石样；4-5天后细胞增殖成片状的细胞团。
供体裸气管制备：HE染色提示,供体气管上皮细胞完全被去除,基底膜完整,软骨组织正常。
上皮细胞灌注及大鼠气管异位移植模型建立：每例模型建立平均用时5.2±1.5分钟。全组(n=80)受体动物术后无一例死亡,生存率为100%,亦未出现感染等术后并发症。
结论
采用胰酶冷消化法能够安全、彻底去除气管上皮细胞,而对其它组织基本无损伤；改进型无血清完全型F12培养方法体外培养上皮细胞培养效果良好,是一种相对简单、实用、有效的培养方法；细胞悬液灌注进行上皮细胞在气管内的种植,操作十分简便,能够很好的保留细胞悬液,为上皮细胞的贴壁、生长和分化创造了条件；大鼠背部皮下气管异位移植模型操作简单、存活率高。
第二部分免疫抑制治疗对受体上皮细胞灌注后大鼠气管异位移植再上皮化的影响
目的
上皮细胞被完全去除之后,气管其它组织依然存活,其中某些细胞和组织也存在有MHC抗原。这也就意味着供体裸气管仍然具有一定的免疫原性,移植后仍然有可能诱发一定程度的免疫排斥反应。而免疫排斥反应是否会对受体源性的再上皮化过程产生不利影响?是否需要实施免疫抑制治疗?免疫抑制治疗的最佳方案是什么?在适当条件下受体上皮细胞灌注后大鼠气管异位移植的最终结果如何?这些问题都需要通过进一步的实验来给予解答。
方法
大鼠气管异位移植模型建立后,将实验动物完全随机分为六组。对照组1(n=10)：供体裸气管内灌入生理盐水,不使用任何免疫抑制剂；对照组2(n=10)：供体裸气管内灌注受体上皮细胞悬液,不使用任何免疫抑制剂；实验组1(n=10)：供体裸气管内灌注受体上皮细胞悬液,移植后受体鼠每日肌肉注射FK506 (0.5 mg/kg/d),连续使用3天；实验组2(n=10)：供体裸气管内灌注受体上皮细胞悬液,移植后受体鼠每日肌肉注射FK506 (0.5 mg/kg/d),连续使用10天；实验组3(n=10)：供体裸气管内灌注受体上皮细胞悬液,移植后受体鼠每日肌肉注射FK506 (1.0 mg/kg/d),连续使用3天；实验组4(n=10)：供体裸气管内灌注受体上皮细胞悬液,移植后受体鼠每日肌肉注射FK506 (1.0 mg/kg/d),连续使用10天。移植后第28天,处死大鼠,取出移植气管,标本分切后分别进行以下检测：组织形态学评价、透射电镜观察、淋巴细胞浸润计数、上皮细胞分化鉴定(CK14、CK18和CFTR)、上皮细胞纤毛比例和气管壁纤维组织含量测定。
结果
实验组的总体情况明显优于对照组。其中,对照组1的结果最不理想,未见任何上皮细胞再生,而且管腔出现严重狭窄、闭塞；对照组2也并未实现再上皮化,但稍好于对照组1；实验组1和实验组2相比,各项检测结果基本相似：气管内壁仅在局部有上皮细胞再生,且细胞尚未分化成熟,从而提示在这两组中存在有受体源性的再上皮化,但结果并不理想；实验组3和实验组4的结果非常相近,同时也是最为理想的：受体源性再上皮化取得成功,气管内覆盖了完整连续的成熟的纤毛柱状上皮细胞,并具有腺体分泌功能、屏障防御功能。
结论
供体裸气管内灌注受体上皮细胞后再实施异位移植,在一定的条件下,能够成功实现受体源性的完全再上皮化；而在这一过程中,仍需进行短期、低剂量的免疫抑制治疗；使用FK506的最佳方案是在移植后连续使用3天,剂量为1.0mg/kg/d;上皮细胞,即便是尚未附壁的上皮细胞,可能对于移植后的阻塞性病变有抑制作用。
第三部分受体上皮细胞灌注后大鼠气管异位移植再上皮化过程的研究
目的
第二部分的实验已经证实,在短期、低剂量的应用免疫抑制剂的情况下,供体裸气管内灌注受体上皮细胞后再实施异位移植,能够成功实现受体源性的完全再上皮化。但是对于其具体的演进过程尚缺乏足够的了解。
方法
大鼠气管异位移植模型建立后,将实验动物完全随机分为六组,移植后所有受体鼠每日肌肉注射FK506 (1.0 mg/kg/d),连续使用3天。在移植后第3天、第7天、第14天、第21天、第28天、第35天,分别随机处死一组大鼠(n=10),取出移植气管,分切后进行以下检测：组织形态学评价、透射电镜观察、上皮细胞分化鉴定(CK14、CK18和CFTR)、上皮细胞增殖能力检测(Ki67)和上皮细胞纤毛比例测定。
结果
在移植后的第3天,气管内壁局部覆盖有无纤毛的扁平上皮细胞；透射电镜发现此处细胞结构正常,偶见有异常短小、稀疏的类纤毛样结构,细胞间隙宽大,未见明显的细胞连接；CK14、CK18和CFTR的表达均处于低水平,Ki67处于高水平,纤毛比例低。
第7天,气管内壁完整覆盖有无纤毛的扁平上皮细胞。其余结果与第3天相似。
第14天,上皮细胞层为扁平细胞和柱状细胞交替,透射电镜发现部分柱状细胞处呈现假复层结构,存在杯状细胞,柱状细胞表面可见少量纤毛,细胞间隙较窄,可见细胞连接；CK14和CFTR的表达仍处于低水平,CK18开始升高,Ki67开始下降,纤毛比例开始升高。
第21天,气管内壁覆盖完整的柱状上皮细胞,偶见刷状缘,透射电镜发现大部分细胞层为假复层结构,其余表现与第14天相似；CK14的表达仍处于低水平,CK18达到峰值,CFTR开始升高,Ki67继续下降,纤毛比例继续升高。
第28天,气管内壁覆盖完整的柱状上皮细胞,可见较明显的刷状缘,透射电镜发现假复层结构明显,可见杯状细胞、腺体分泌细胞、基底细胞等多种细胞类型,细胞间隙狭窄,细胞连接紧密；CK14和Ki67的表达处于低水平,CK18维持高水平,CFTR达到峰值,纤毛比例达到峰值。
第35天的表现与第28天相仿。
结论
受体上皮细胞灌注后大鼠气管异位移植再上皮化的演进过程为：灌注后细胞迅速附壁,其中成功附壁的上皮细胞开始增殖,进而完整覆盖气管内壁,形成单层扁平上皮组织,而后上皮细胞开始分化,最终成为成熟的假复层纤毛柱状上皮组织。
Background
In the end-to-end anastomosis of trachea, the resectable length is restricted to 1/2 of the total length (6 cm) in adults or 1/3 in children, and replacement of longer sections will only be feasible if a safe and functional tracheal replacement can be developed, such as tracheal allografts, artificial or tissue-engineered airway, autogenous tissues graft and so on. Among them, tracheal allografts may be the most effective option for achieving airway reconstruction at present. Although more and more researchers and clinicians focus on tracheal transplantations, there are a few clinical reports of tracheal allotransplantations. And immune rejection is one of key obstacles in tracheal allografts. Immunosuppressive therapy increases the possibility of sever infection and malignant tumor, while improving the allografts. Therefore it is necessary to find an alternative to suppress the immune rejection after tracheal transplantation.
As is known, the immune rejection after allografts can be suppressed by eliminating the antigenicity of grafts. It was reported that tracheal antigenicity may be eliminated or reduced by the removal of epithelium in trachea. However, epithelial tissues play a key role in functional reconstruction and transplantation of trachea. Therefore, it is the best way to promote reepithelization from the recipient after the removal of donor epithelium.
Heretofore, it is rare that the study of reepithelization from the recipient after the removal of donor epithelium in tracheal allografts. Thus the key steps in reepithelization process are not clear, the technique means are imperfect, and the theoretical basis is very poor. We plan to use enzymatic dissociation with trypsin to remove the epithelium from the rat donor trachea which is then colonized by epithelial cells that have been cultured from cells taken from the recipient. Afterwards, the donor trachea is heterotopically transplanted to the dorsum of recipient rat and reepithelization will be completed. And our objectives are to objectively evaluate the present method, to detailedly describe the whole process and to optimize the technique means.
PART ONE
Culture and perfusion of epithelial cells from rat trachea, production of donor nude trachea and establishment of heterotopic tracheal transplantation model
Objective
We plan to perfuse the epithelial cells cultured from the recipient trachea into the donor nude trachea in which donor epithelial tissues has been removed by enzymatic dissociation in order to reduce the antigenicity of donor trachea and complete reepithelization from the recipient. It is the key steps that culture and perfusion of epithelial cells from rat trachea, production of donor nude trachea and establishment of heterotopic tracheal transplantation model.
Methods
The tracheal epithelial cells of SD rats were primarily cultured in Ham's F-12 and the cultured cells were diluted to cell suspension (10×106 cells/ml).
Wistar rats (n=80) were taken as donors and SD rats (n=80) as recipients. Tracheas (1.5-2.0 cm) were excised from below the larynx to the major bronchi. The explanted tracheas were digested for 18 h with 0.25% trypsin at 4℃, then the qualification of donor nude tracheas were evaluated by HE stain. The qualified donor nude tracheas were stored in Ham's F-12. One end of donor nude trachea was ligated, and the recipient cell suspension was perfused into its lumen before ligation of another end. Afterwords, donor tracheas were heterotopically subcutaneous transplanted to the dorsum of recipients.
Results
Epithelial cells being round-shape have good diopter and suspended in culture solution. After 24 hours, the majority of cells started to adhere to the wall. After 3 days, cells showed typical "flagstone" appearance. After 4-5 days, cells formed the flake-shape cell clusters. HE stain showed that epithelial cells were totally removed from the donor trachea with integrated basement membrane and normal cartilage tissues. The mean time of animal model establishment is 5.2±1.5 min. There was no postoperative complications, eg. infection, and the survival rate was 100%.
Conclusion
The enzymatic dissociation with trypsin is a safe and effective method to remove epithelial cells from the donor trachea. Ham's F-12 is a suitable culture solution for our study. Perfusion of cells suspension is a simple and available technique to complete the epithelial implantation. Because of easy operation and high success rate, heterotopic subcutaneous tracheal transplantation model in rat is the best choice in present study.
PART TWO
Effect of immunosuppressive therapy on reepithelization after rat heterotopic tracheal graft with recipient epithelial cells perfusion
Objective
After totally removal of epithelial cells, there are still MHC antigens existing in rest cells and tissues, which means donor nude trachea still has antigenicity and immune rejection will take place after transplantation. And we need to answer the following questions:Will immune rejection has adverse influence on reepithelization from the recipient? Is it necessary to perform the immunosuppressive therapy? What is the best treatment regimen of immunosuppressive therapy? How is reepithelization after rat heterotopic tracheal graft with recipient epithelial cells prefusion?
Methods
After establishment of heterotopic subcutaneous tracheal transplantation model in rat, animals were randomly divided into six groups. Control group 1 (n=10):normal saline was perfused into donor nude trachea, without immunosuppressive therapy; Control group 2 (n=10):recipient epithelial cells suspension was perfused into donor nude trachea, without immunosuppressive therapy; Test group 1 (n=10):recipient epithelial cells suspension was perfused into donor nude trachea, with intramuscular injection of Tacrolimus (0.5 mg/kg/d) only for three consecutive days after transplantation; Test group 2 (n=10):recipient epithelial cells suspension was perfused into donor nude trachea, with intramuscular injection of Tacrolimus (0.5 mg/kg/d) for seven consecutive days after transplantation; Test group 3 (n=10):recipient epithelial cells suspension was perfused into donor nude trachea, with intramuscular injection of Tacrolimus (1.0 mg/kg/d) only for three consecutive days after transplantation; Test group 4 (n=10):recipient epithelial cells suspension was perfused into donor nude trachea, with intramuscular injection of Tacrolimus (1.0 mg/kg/d) for seven consecutive days after transplantation. All rats were killed on 28 days after transplantation and then the implanted tracheas were harvested. In all specimens, histological and ultrastructural changes were evaluated, infiltrated lymphocytes were counted, differentiation of epithelium (CK14, CK18, CFTR) were identified, percentage of cilia and fibrosis tissues were calculated.
Results
Generally the results of test groups were better than control groups. In the latter, there was not any epithelial regeneration, and tracheal stenosis, even lumen occlusion, were found. Test group 1 and test group 2 had the similar results that immature epithelial regeneration took place in the regional area of tracheal inner surface. And test group 3 and test group 4 also had the similar results that mature ciliated columnar epithelium covered the whole inner surface of trachea.
Conclusion
It is an effective way to complete reepithelization from the recipient after rat heterotopic tracheal graft with recipient epithelial cells injection. And short-term and low dosage immunosuppressive therapy is necessary. The best regimen is the intramuscular injection of Tacrolimus (1.0 mg/kg/d) for three consecutive days after transplantation. Moreover, epithelial cells from the recipient may inhabit the development of obliterated airway disease.
PART THREE
Study on the process of reepithelization after rat heterotopic tracheal graft with recipient epithelial cells perfusion
Objective
In part two, we have improved that it is an effective way to complete reepithelization from the recipient that heterotopic tracheal graft with recipient epithelial cells perfusion in rat with short-term and low dosage immunosuppressive therapy. But it is still unclear about the detailed process of reepithelization.
Methods
After establishment of heterotopic subcutaneous tracheal transplantation model in rat, the animals were randomly divided into six groups. All animals were intramuscular injected of Tacrolimus (1.0 mg/kg/d) only for three consecutive days after transplantation. Rats (n=10) were killed on 3,7,14,21,28 and 35 days respectively after transplantation and then the implanted tracheae were harvested. In all specimens, histological and ultrastructural changes were evaluated, differentiation (CK14, CK18, CFTR) and proliferation (Ki67) of epithelium were identified, percentage of cilia were calculated.
Results
On 3 days after transplantation, the regional areas of tracheal inner surface were covered by non-ciliated flate cells with normal cellular ultrastructure and wide intercellular space, but without cell junctions. And the levels of CK14, CK18, CFTR and percentage of cilia were low, while Ki67 was high.
On 7 days, the whole areas of tracheal inner surface were covered by non-ciliated flate cells. And the rest results were similar to those on 3 days after transplantation.
On 14 days, the whole areas of tracheal inner surface were covered by the mixture of flate cells and columnar cells with sparse cilia. The latter arranged in pseudostratified structure which consisted of goblet cells, basal cells and so on. Cell junctions were found under Transmission Electron Microscope. The levels of CK14, CFTR were still low, but the level of CK18 and percentage of cilia started to increase and the level of Ki67 started to decrease.
On 21 days, the whole areas of tracheal inner surface were covered by columnar cells. The majority of cells arranged in pseudostratified structure. The level of CK14 was still low, and percentage of cilia still increased and the level of Ki67 decreased. But the level of CFTR started to increase and CK18 reached the peak level.
On 28 days, the whole areas of tracheal inner surface were covered by ciliated columnar cells arranged in pseudostratified structure. The narrow intercellular space and tight cell junctions were observed under Transmission Electron Microscope. The levels of CK14, Ki67 were low, and the levels of CK18, CFTR and percentage of cilia were high.
The results on 35 days after transplantation were similar to those on 28 days.
Conclusion
Cells adhered to tracheal inner wall quickly after perfusion of cells suspension. Then the adherent flate cells started to proliferate until the whole inner surface was covered. And the cells started to differentiate. Finally, mature pseudostratified ciliated columnar cells appeared.
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