促进异种无细胞真皮血管化和体外构建复合皮移植的实验研究.pdf

(2)在ADM表面种植成纤维细胞，测定培养上清中IL一6、IL一8、TGF- pl 及细

胞外基质层粘连蛋白、透明质酸的浓度；采用RT—PcR检测bFGF、aFGF、KGF

mRNA表达：观察移植早期血管化速度；以特异性寡核苷酸探针杂交，追踪

移植后成纤维细胞的转归。

(3)以ADM为载体，构建含表皮细胞成纤维细胞的复合皮，移植于裸鼠，观察创

(4)按1：1、l ：3、1：5的比例，将Bal b／c小鼠(自体)表皮细胞与人(异种)表皮细

胞混合培养形成复合皮，移植于小鼠全层皮肤缺损创面，观察免疫排斥与

(5)观察转EGF基因人表皮细胞的生物学活性，将Bal b／c小鼠表皮细胞混合转基

因人表皮细胞培养形成复合皮，移植后检测EGF的分泌，观察基底细胞

PCNA(增生陛细胞核抗原)阳性表达率。

(6)将ADM与自体大张刃厚皮片、网状皮、邮票样皮片等复合移植，应用于临床，

观察存活率，从外观、功能等方面评估创面愈合质量。

(1)微孔ADM可增强渗透性，有利于移植早期新生血管形成，密集的微孔结构在

移植后2周左右，基本被新生结缔组织填充，无明显瘢痕样结构形成，并

不影响创面愈合质量。与自体皮片复合移植，存活率为(89．5+6．0)％，明

显高于无孔ADM组(63．2+7．8)％。

(2)在ADM表面种植成纤维细胞培养，可分泌IL一6、IL一8、TGF—B1及层粘连

蛋白、透明质酸，检测到KGF、bFGF、aFGF mRNA表达。移植后成纤维细胞

存活、增殖，并较长时间存在。与未种植成纤维细胞的ADM相比，移植后l 一2

周，真皮浅层微血管数目明显增多，创面收缩率减小。

(3)ADM无明显毒性，表皮细胞可在ADM表面生长、增殖，形成细胞膜片，但粘附

种植一定量的成纤维细胞“修饰”ADM表面，可增强表皮细

胞对ADM的粘附，促进生长、增殖，有利于提高移植存活率。

(4)单纯人表皮细胞复合皮移植于Bal b／c小鼠，3周左右被排斥，而Bal b／c表

皮细胞混合人表皮细胞移植后创面无明显裸露，免疫组化提示人表皮细胞

逐渐被“排挤”到表皮外层，最终消失。移植后4周，新生表皮、真皮及基

(5)转EC- F基因表皮细胞经G418筛选后，可稳定分泌一定量的EGF(8．3pg／ml 。11．3

pg／m] )，且表皮细胞具有的基本生物学性状无明显改变。与小鼠自体表皮细

胞混合，移植后l 、2周内可表达EGF，与单纯小鼠表皮细胞复合皮移植相

比，基底层中PCNA阳性细胞数增多。

(6)微孔ADM与患者自体大张刃厚皮片、网状皮、邮票样皮片等复合移植，存活

率较高。创面愈合后色素沉着浅，外观平整、光滑，质软，无明显瘢痕形

(1)在ADM上打孔可增强渗透性，提高与自体皮片复合移植的存活率；而且并不

(2)成纤维细胞易于获取，培养方便，在ADM支架表面种植成纤维细胞简便易行，

可形成具有较高生物学活性的真皮替代物，促进移植后早期血管化，控制

创面收缩，为含表皮细胞复合皮的构建与移植奠定了基础。

(3)ADM无明显毒性，可于体外构建含表皮细胞成纤维细胞的复合皮，并封闭深

(4)自体／异种表皮细胞按一定比例混合移植，能形成新的上皮。

(5)转EGF基因人表皮细胞经G418筛选，可稳定分泌EGF，与小鼠自体表皮细胞

混合后移植，在移植早期分泌EGF，并促进细胞增殖。

(6)微孔ADM与自体皮片复合移植，存活率较高，可提高烧伤患者创面修复质量。

关键词烧伤：创面修复；无细胞真皮；复合皮：基因转染；皮肤组织工程

鱼00‘：rae e

Improvement onvascul ari zat i on oft he acel l ul ar xeno· dermi s

and t ranspl ant at i on oft he composi t e ski n recombi ned／n vi tro

Background

Cl osure of ful l - thi ckness ski n defect s i s i mport ant

t o t he t reat ment of pati ents wi t h

bum，traumati c i nj ury, chroni c ski n ul cer

or hypert hopi c scar．The present

approach i s

grafti ng aut ol ogous ski n，whi ch oft en resul ts i n heavy pi gment at i on and scarri ng i n donor

si tes，and t he amount of ski n obt ai ned by thi s met hod i s l i mi ted for pati ents wi th l arge area

ski n inj uries．The t echni que of ski n ti ssue engi neeri ng has been a newmet hod t o provi de

adequat e ski n grati s andi mprove wound heal i ng qual i ty．

Prepari ng t he dermal repl acement and reconsti tuti ng t he composi t e ski n i n vi t ro i s t he

t wo mai n aspect s

of ski n t i ssue engi neeri ng．The dermal

substi tutes

graft ed wi t h an

overl yi ng t hi n aut ol ogous

ski n coul d exhi bi t

an excel l ent

cosmet i c appearance and

funct i onofwound cl osure．On t he ot her hand，the composi t e ski n recombi ned i n vi t ro coul d

provi de l arge amount &ski n gFai Rs for pat i ent s wi th ext ensi ve ski n l oss．

devel opment

repl acement

parti cul ar

engi neeri ng．Vari ous dermal anal ogs such as hi tegra, Al l oderm，Dermagraft et al have been

devel oped and used as dermal t empl at e t o cover full—thickness wounds successfully．The

dermal repl acement grafted wi t h thi n aut ol ogous ski n coul d short en heal i ng ti me of donor

reci pi ent

funct i on

pi gmentati on，wound conVacfi on and beaer durabi l i ty．However，these dermal

substi tutes

deri ved fromal l ogenei c ski n have l i mi ted SOurCeS as wel t

as sl owvascul ari zat i on or poor

graft ed si mutaneousl y、Ⅳi t}l

autografts．Therefore．devel opment

of economi cal

dermal repl acement wi tl l good qual i ty i s necessary for t he management ofbumpati ents．

The composi t e ski n recombi ned加vi t ro coul d serve as

a source of ski n grafts．The

arti fi ci al ski n compri sed ofaut ol ogous kerat i nocyt es cul t ured i n vi t ro and porous col l agen

sponge membrane has been graft ed ont o the exci sed bumwounds．However, t here i s some

quest i on such as t he l ong cul ture ti me，l ow anti bacteri al capaci t y and poor t ake rat e that

restri cts t he use of ski n equi val ent ．The proposed key reasons i ncl ude poor di ffusi on of

nutri ents，l ack of ready· formed capi l l ary network and sl owneovascul ari zati on．The dat um

have shown that t he most l i kel y mechani smfor ski n graft survi val

i s di ffusi on ofnutri ents，

fol l owed by i noscul afi on of t he reci pi ent bed capi l l ari es t o t he severed ends of t he graft

vessel s and neovascul ari zati on．It i s di ffi cul t for t he nut ri ent s i n pl asma t o pass t hrough the

Al l oderm，a

acel hi l ar

al l o—dermal

mat ri x，because

effecti ve

mi crohol es

responsi bl e for the di ffi asi on of l i qui d．0n t11e ot her hand, t he composi t e ski n does not

cont ai n capi l l ari es．So epi dermi s coul d not t ake enough nut ri t i on unti l t he establ i shment of

capi l l ary vascul ar fl owduri ng weeks

t o 2 postgraff，al l t hese l ead t o t he graft sl ough and

dead．Therefore it i s essent i al for t he composi te ski n survi val

t o nurt ure t he kemt i nocyt es

seeded on t he surface ofdermal substi tute．

In recent years．t11e st udy on ski n ti ssue engi neeri ng at home has beenmade and i s sti l l i n

progress．The acel l ul ar dermal mat ri x( ADM) has been prepared，but t he same quest i on i s

l ow t ake

rate．It’s repoaed about

t he reconsti tuti on of composi t e ski n that

al l ogenei c

kerat i nocyt es seeded on t he surface of coHagenmat ri x graf【ed ont o t he bumwounds after

exci si on ofeschar．Al l ogenei c cel l s woul d be rej ected i n t he end i nspi te ofl owanti geni ci ty

after cul ti vated i n vi tro．Therefore，al l ogenei c kerat i nocyt es coul d not be used as permanent

coverage．Howt o i mprove the poor engraft rat e of ADMwi t h an overl yi ng aut ol ogous

ski n，and t o reconsti tute t he composi t e ski n composed t he ADMas vehi cl e i s t he quest i on

we must answer．

Ont he basi s oft he present progress，0121 st udy i s desi gned t o i mprove penet mt i ng abi l i ty

of ADMand prol i ferati ve capaci ty of kerat i nocyt es cul t ured on t he dermal mat ri x．These

i ncl ude：

(1)To i mprove t he penet mt i ng abi l i ty ofADM．ADMdoes not cont ai n mi crohol es that are

penet rat i ng

overl yi ng

kemt i nocyt es

al i ve．Regul ar mi crohol es punched mechani cal l y on t he ADMcoul d promot e the

di ffusi on of nut ri ent s through grafts due t o t he reason of i mbi bi t i on．They coul d al so

prevent fl ui d，bl ood，gas fromcol l ecti on benet h gral ts．

accel erat e

vascul ari zat i on of ADMafter

transpl antati on．Fi brobl asts

wi t h l ow

anti geneci ty, excel l ent bi ol ogi cal

vi abi l i ty coul d secret many ki nds of cyt oki ne and

growt h fact or, whi ch i mprove the

growth，prol i ferati on of endot hel i al

format i on oft he capi l l ary vessels．Therefore，the l i vi ng dermal repl acement consi sti ng of

ADMand fi brobl asts woul d resul t i n rapi d vascul ari zati on．

(3)To promot e t he prol i ferat i on of kerati nocytes．It’s known that EGF coul d promot e

kerat i nocyt es growt h．Cl osure wound wi th aut ol ogoas kerat i nocyt es mi xed wi th EGF

gene transferred al l o(xeno)genei c

save autografts，meanwhi l e, the

transferred cel l s coul d promot e autol ogous ski n generat i on by secreti ng EGF．In t he

end，al l ogenei c kerat i nocyt es woul d be rej ected，whi ch coul d avoi d t he potenti al danger

t o humanbei ng resul t i ng fromgene t herapy

obj ecti ves

(1)To promot e engraft ment

of ADMwi t h overl yi ng autografts

by i mprovi ng t he

i mbi bi t i on and vascul afi zat i onofADMpostgraff．

fl ai l —thi ckness

wi t h the‘sandwi sh’composi te

ski n graft

compri sed cul t ured kemt i nocyt es and fi brobl asts and ADM．

(3)To eval uat i on t he effect of wound heal i ng i n mi ce model after t ranspl ant at i on of t he

composi t e ski n，whi ch composed ofBal b／c and humankerati nocytes(gene t ransferred

kerati nocytes)cul tured onADM．

Mat eri aI&Met hods

( 1) ADMwas punched mechani cal l y t o produce regul ar hol es from500¨ mt o 800p．mi n

di ameter，separated by a di st ance of 3mmt o 5mm．Then, t he penet rat i ng funct i on and

t ake rat e ofADMgraRed、Ⅳi t}1 overl yi ng aut ol ogous ski ns were eval uated．

(2)The supemat ant of fi brobl asts cul t ured on t he dermal si de ofADMWas col l ect ed for

ri se，three

di fferent

pept i des

(i nterl i uki n一6，i nterl euki n-8，and

t ransformi ng growt h factorl 3 1)and t wo di fferent extracel l ul ar matri x(1ami ni mand

hyal uroni c aci d)were determi ned．The mRNA expressi on of aFGEbFGEKGF i n

fi brobl asts Was det ect ed by usi ng reversi bl e t ranscri pt i on PCRtechni que．nl e fate of

fi brobl asts seeded on ADMafter thanspl antati on Was t raced by usi ng i n si tu hybri

di zat i on wi mspeci es- speci fc DNAprobes．

f3、The composi t e ski ns were reconsti tuted wi mkerafi nocyt es and fi brobl asts cul t ured on

t he surface of ADM，then，the grafts were appl i ed t o the ful l

t hi ckness wounds i n

at hymi c mi ce model ，and t he heal i ng effect Was assessed．

(4)Bal b／c mi ce and human kerat i nocyt es were cocul t ured on ADMat t he fol l owi ng

rati os．1：1．1：3

1：5．The resul t i ng composi t e grafts were transpl anted ont o t he full—

thi ckness wounds

i n BaLb／c

mi ce，the i mmune rej ecti on of

grafts and t he

regenerat ed epi dermi s Was observed

(5)The bi ol ogi cal vi abi l i ty of EGF gene gransferred kerat i nocyt es Was eval uated．ADM

seeded wi th Bal b／c

kerat i nocyt es and gene

gransferred human kerat i nocyt es

di fferent rati os was transpl anted t o t he ful l -thi ckness wounds．Then，EGF secret ed by

human kerat i nocyt es was detected．PCNA(prol i ferati ng cel l nucl ear anti gen)- posi ti ve

cel l s i n basal l ayer oft he regenerat ed ski n were counted．

( 6) ADMwas t ranspl ant ed t O t he ful l —thi ckness defects wound i n burn pati ents，then the

aut ol ogous ski n grafts were pl aced over ADM．The t ake rat e of grafts and cosmet i c

appearance，funct i on oft he regenerat ed ski n were assessed．

(1)Mi crohol es punched on ADMwere responsi bl e for t he penet rat i ng funct i on and

vascul ari zati on．Granul ati on ti ssue

fi l l ed t he hol es

i n ADMabout

　　2 weeks

transpl antati on．The engraft ment rate of the aut ol ogous ski n pl aced over ADMwi t h

hol es was( 89 5±6．O)％，whi ch i s si gni fi cantl y hi gher t han di d ADMwi t hout hol es．Tk

regenerat ed ski n charact eri zed wi th good durabi l i ty and el asti ty had more smoot her

and l ess el evat ed surface．

(2)Fi brobl asts seeded on ADMcoul d secret growt h pept i des IL- 6，IL一8 and TGF—p1 as

as extracel l ul ar mat ri x hyal uroni c aci d and l ami ni n，t}l ey coul d express mRNA

of aFGF, bFGF and KGF too．Fi brobl asts postgraff coul d survi ve and prol i ferate．n他

newbl ood vessel s i n superfi ci al l ayer of ADMwi th fi brobl asts were more t han i n

ADMwi thout

fi brobl asts duri ng l to 2 weeks postsurgery．Fi brobl asts coul d i nhi bi t

血e wound contracti on．

( 3) ADM showed

cyt ot oxi c

acti vi ty．Kerati nocytes

on ADM coul d

grow, prol i ferate and reach confl uence，but i ts at t achment t O t he surface of ADMwas

poor．Fi brobl asts

and promot e t he

kerat i nocyt es

attachment幻ADM．as wel l as t he survi val postsurgery．

r4、111e composi t e ski n onl y cont ai ni ng human kerat i nocyt es coul d not cl ose t he wound

permanent l y i n Bal b／c mi ce model due t o i mmune rej ecti on However，t he composi t e

ski n cont ai ni ng human and

kerat i nocyt es

cover t he wound．The

i mmunohi stol ogi cal anal yses showed，human keat i nocyt es l ocat ed t he upper l ayers of

t he newl y generat ed cut aneous t i ssue．and di sappeared at t he end．The generat ed

epi dermi s，dermi s and basal membrance exhi bi t ed wel l —organi zed st ruct ure．

( 5) EGF gene transferred kerat i nocyt es sel ected wi th G418 coul d secret EGF(8．3pg t o

11．3p∥ml supematant)．EGF coul d be det ect ed i n t he epi dermi s generat ed after t he

chi meri c i mpl antati on for 1 t o 2 weeks postgraff．The number of PCNA—posi t i ve cei l s

i n t he base l ayer ofepi dermi s si gni fi ci antl y i ncreased．

f6)The hi ghl y porous ADMcombi ned wi th aut ol ogous ski n exhi bi t ed good survi val ．The

si tes recei vi ng ADMhad i ncreased el asti ci ty and durabi l i ty, a smoot her surface and

l i ght pi gment at i on as compared t o t he control sites．

Conel usi ons

(1)The porous ADMhad excel l ent fl ui d penel rance and support ed t he survi val of

ovel yi ng autografl s．

(2)Fi bmbl ast s

coul d promot e vascul azat i on of ADMi n t he

i mpl antati on, and i nhi bi t wound contracture．Thi s woul d benefi t t he reconsti tuti on

and transpl antati onoft he composi t e ski n．

( 3) ADMsupport ed t he cul t ured kerat i nocyt es and fi brobl asts’growth，prol i ferati on．The

composi te ski n grafts cont ai ni ng keral i nocyt es and fi bmbl ast s coul d permanent l y

cl ose t he deep wound．

(4)The chi meri c ski n graft s of cocul t ured aut ogenous and xenogenous kerat i nocyt es

combi ned wi t h ADMvcere a useful l approach for t he cl osure of ful l - thi ckness

( 5) EGF gene t ransfect ed human kerat i nocyt es sel ected by G41 8 coul d secret EGF

stabl y．When t he human kerat i nocyt es t oget her wi t h mi ce ones were i mpl anted

ont o t he wound，EGF secret ed by human kerat haocyt es

coul d promot e cel l s

prol i ferati on．

(6)The porous ADMcombi ned wi th aut ografl s exhi bi t ed a hi gh percent age t ake and

i mproved t he qual i ty ofwound heal i ng．

wound heal i ng

acel l ul ar dermal mat ri x

composi t e ski n

gene transfer

ski n ti ssue engi neeri ng

烧伤是一种常见的损伤，平时与战时发生率均较高。为大面积深度烧伤患者

提供皮源提高救治率和改善中小面积烧伤患者创面修复质量，提高康复质量与生

存能力，对战时及平时成批烧伤患者的救治具有重要的现实意义。

近年来，皮肤组织工程技术的应用为解决皮源不足、提高创面修复质量提供

了新途径。皮肤组织工程主要包括两个基本的方面，即研制真皮支架和体外构建

复合皮。其中真皮支架的研制既是基础也是技术关键。国外研究者已成功地开发

了多种真皮支架，如来源于尸体皮的异体无细胞真皮替代物A1] oderm等。结合

自体大张刃厚皮片、网状皮已成功地用于深度烧伤创面的修复，可减少供皮区色

素沉着、瘢痕形成的机率。受皮区愈合后外观平整、光滑，无明显点状、网状瘢

痕，富有弹性“’。国内的研究者在无细胞真皮替代物的研制方面亦取得了一定的

进展。一些研究单位采用猪皮，经脱细胞处理后制成异种无细胞真皮替代物，并

应用于临床。但异种无细胞真皮复合自体皮片一步移植时，存活率较低”] 。在复

合皮研究方面，已将表皮细胞、成纤维细胞分别种植于胶原膜或聚乳酸／聚羟基

乙酸网两面，经体外培养形成复合皮移植，并初应用于临床，可减少自体皮需求

量，提高救治率。’”。组织工程技术的开发与应用为烧伤、创伤、慢性皮肤溃疡、

瘢痕整形等的治疗带来了前所未有的希望。但无论是真皮替代物结合自体皮片还

是种植表皮细胞形成复合皮移植，都存在许多与实际应用密切相关的问题，大大

限制了临床应用。如A1] oderm来源于尸体皮，取材有限，价格昂贵，而且存在

细菌、病毒污染的问题。真皮替代物复合自体皮片一步移植，存活率较低。而以

真皮支架为载体的复合皮移植，抗感染能力差，应用于大面积烧伤患者切痂创面，

存活率仅50％左右。主要原因是由于真皮支架渗透性差，血管化速度相对较慢o] 。

研究表明⋯’，皮片植入创面后其营养来源按发生先后分别为创面渗液、血管网吻

合、新生血管形成。无细胞真皮尽管具有去除毛发、汗腺、皮脂腺、微血管后残

存的腔隙，但孔隙较少，且并不贯通整个真皮基质。因此植入创面后，创面基底

血浆等渗液难以透过无细胞真皮，不能维持其上覆盖的皮片或表皮细胞早期(1。2

天)的营养需要。无细胞真皮移植后，新形成的毛细血管需1～2周才能达到真皮

浅层。这些因素均可导致移植早期覆盖于真皮替代物上的自体皮片或种植的表皮

细胞营养供应不足，增殖缓慢，甚至死亡脱落。因此，改善真皮替代物的渗透性

及血管化速度是提高移植存活率的关键所在。

本课题拟从改善真皮替代物渗透性、提高复合皮中表皮细胞自身的增殖活力

等方面入手进行初步探讨，具体包括以下几方面：(1)改善真皮替代物渗透性。

在无细胞真皮替代物上打密集微孔，根据毛细管原理，创面基底血浆等渗液可沿

管壁渗透到真皮表面，且可防止移植后皮下积液、积血、积气。医I其孔径较小，

与自体刃厚皮、网状皮复合移植，可避免形成明显的点状或网状疤痕。(2)促进

无细胞真皮血管化。成纤维细胞抗原性弱，生物活性高，可分泌多种细胞因子，

如IL一6、IL一8、TGF- - [ 3l 、aFGF、bFGF及KGF等。体外实验表明，将成纤维细

胞与表皮细胞混合培养，前者通过释放上述多种生长因子、细胞因子促进后者生

长、增殖。将血管内皮细胞混于胶原凝胶中培养，并不能形成血管样结构，而当

引入成纤维细胞后，内皮细胞增殖活性明显增强，并诱导内皮细胞聚集，形成明

显微血管样管腔。动物实验也表明，成纤维细胞可促进创面表皮化，加速微血管

形成”】。因此，在无细胞真皮表面种植成纤维细胞，形成活性真皮替代物，可望

促进移植后血管化速度。(3)提高表皮细胞增殖活力。一定浓度的EGF可促进表

皮细胞增殖。我们已构建EGF基因表达载体并转染正常人表皮细胞，移植后可分

泌EGF，促进创面愈合。如果将转EGF基因的异体表皮细胞与自体表皮细胞混合

后培养，形成细胞膜片移植，不但节省自体皮源，而且转基因细胞不断分泌生长

因子，促进表皮细胞增殖。异体细胞最终将被排斥，可避免因基因治疗带来的潜

在危害。本实验拟以Bal b／c小鼠为受体，将转EGF基因的人表皮细胞与小鼠自

体表皮细胞按一定比例混合种植于无细胞真皮表面，封闭小鼠全层皮肤缺损创

面，观察对创面的修复作用。

1．Wai nwri ght DJ．Use of

an aeel l ul ar al l ograft

mat ri x( A1l oderm) i n

t．he management

of fMl - t hi ckness burns．8urns，1995，21：243—248

2．冯祥生，潘银根，谭家驹，等．异种(猪)脱细胞真皮与自体表皮复合移植研究．中

华整形烧伤杂志，2000，16(1)：40—42

3．Compt on CC，But l er

a1．Organi zed ski n st ruct ure

regenerat ed

from col i agen—GAG mat ri ces

seeded wi t h aut ol ogous

kerat i nocyt es J

Dermat ol ，1998，110：908—916

4．Hmmbrough JF，Morgan几，Greenl eaf

a1．Composi t e

kerafi nocyt es

a pol y羽acti n mesh· cul t ured

fi brobl ast

substi tute

fi mct i on as a bi l ayer ski n repl acement i n ful l —thi ckness wounds onat hymi c mi ce．J Bum

Care Rehabi l ，1993，14：485- 494

　　5 Pandi t

A．Ashar RFel dman D et

a1．1nvesti gafi on of aci di c fi bmbl ast growt h factor

del i vered t hrough a col l agen scaffol d for t he t reat ment offul l —thi ckness ski n defect i n a

rabbi t model ．Pl ast Recol l St rSurgery, 1998，101：766—775

6．Young DM，Greul i ch KM，Wei er HG，el a1．Speci es—speci fi c i n si tu hybri di zat i on wi t h

fl uorochrome—l abel ed DNAprobes to st udy vascul ari zafi on of human ski n grafts on

at hymi c mi ce．J BumCare Rehabi l ，1 996，1 7：305G1 0

7．Hansbrough JF，Morgan J, Greenl eaf G，et aLDevel opment of a t emporary l i vi ng ski n

repl acement composed of human fi bmbl ast s cul t ured i n Bi obrane，a syntheti c dressi ng

materi al ．Surgery, t994．115：633—637

微孔异种无细胞真皮的研制

无细胞真皮(acel l ul ar

matri x，ADM)与自体皮片复合移植已越来越多

地应用于深度皮肤缺损创面的修复。复合移植最大的缺陷之一是皮片存活率较低。

国内有作者报道⋯，将厚约0．3mm～O．5mn的ADM与自体刃厚皮片重叠一步移植，存

活率只有(60．0±3．1)％。尽管移植ADM可减少供皮区瘢痕形成与色素沉着机会，改

善受皮区创面愈合后的柔韧性与弹性，但较低的存活率大大限制了ADM的推广应

用。为解决这一问题，将ADM拉网(如l ：1)，然后再重叠自体网状皮移植，网孔间

相互错开，适当加压包扎，使自体皮片通过ADM网孑L与创面基底接触。由于网孔较

大，创面愈合后仍可形成网状或点状瘢痕，降低了ADM作为真皮支架的应用价值。

本实验通过在ADM上行贯穿性密集而规则的打孔，改善渗透性与血管化速度，

达到提高移植存活率的目的。

　　1材料与方法

1．1微孔异种ADM的制作

按本实验室建立的方法制备异种(猪)ADM。切取新鲜小猪断层皮片，厚约

0．4mm—O．6mm，消毒后在无菌条件下浸泡于高渗盐水中24小时，撕除表皮。然后

以去垢剂浸泡，去除真皮层中细胞成份。经光镜及透射电镜检测无细胞及细胞碎屑

存在，胶原结构完整。采用机械打孔的方法，在ADM上行规则的密集的贯穿性打孔。

孔径为500¨ m~800“m，孔间距为3rml ～5mm。将制备的微孔ADM裁剪成适当大小，40C

1．2 ADM皮下埋藏试验

雄性sD大鼠21只，(上海西普尔一必凯实验动物有限公司提供)体重200±209。

腹腔注射3％戊巴比妥钠(35mg／Kg体重)麻醉。剃除背部毛发，消毒皮肤，沿背部正

中线切开皮肤、皮下组织。沿切口向腹部两侧分离皮下组织，去除深筋膜直达肌肉

层，充分止血。长条形ADMl cmX4cm大小，以长边中点连线为界，一半打孔，一

半不打孔。以脊柱为分界线，用3- 0手术缝合线将ADM缝合固定于肌肉层上，脊柱

右侧为ADM打孔的部分，左侧为未打孔的部分。再以1号线缝合背部皮肤切口，无

菌纱布覆盖，包扎，适当固定，定期换药，l O天左右拆线。

分别于手术后3天、l 周、2周任选7只SD大鼠，麻醉后切开背部皮肤及皮下

微孔异种无细胞真皮的研制

无细胞真皮(acel l ul ar

matri x，ADM)与自体皮片复合移植已越来越多

地应用于深度皮肤缺损创面的修复。复合移植最大的缺陷之一是皮片存活率较低。

国内有作者报道⋯，将厚约0．3mm～O．5mn的ADM与自体刃厚皮片重叠一步移植，存

活率只有(60．0±3．1)％。尽管移植ADM可减少供皮区瘢痕形成与色素沉着机会，改

善受皮区创面愈合后的柔韧性与弹性，但较低的存活率大大限制了ADM的推广应

用。为解决这一问题，将ADM拉网(如l ：1)，然后再重叠自体网状皮移植，网孔间

相互错开，适当加压包扎，使自体皮片通过ADM网孑L与创面基底接触。由于网孔较

大，创面愈合后仍可形成网状或点状瘢痕，降低了ADM作为真皮支架的应用价值。

本实验通过在ADM上行贯穿性密集而规则的打孔，改善渗透性与血管化速度，

达到提高移植存活率的目的。

　　1材料与方法

1．1微孔异种ADM的制作

按本实验室建立的方法制备异种(猪)ADM。切取新鲜小猪断层皮片，厚约

0．4mm—O．6mm，消毒后在无菌条件下浸泡于高渗盐水中24小时，撕除表皮。然后

以去垢剂浸泡，去除真皮层中细胞成份。经光镜及透射电镜检测无细胞及细胞碎屑

存在，胶原结构完整。采用机械打孔的方法，在ADM上行规则的密集的贯穿性打孔。

孔径为500¨ m~800“m，孔间距为3rml ～5mm。将制备的微孔ADM裁剪成适当大小，40C

1．2 ADM皮下埋藏试验

雄性sD大鼠21只，(上海西普尔一必凯实验动物有限公司提供)体重200±209。

腹腔注射3％戊巴比妥钠(35mg／Kg体重)麻醉。剃除背部毛发，消毒皮肤，沿背部正

中线切开皮肤、皮下组织。沿切口向腹部两侧分离皮下组织，去除深筋膜直达肌肉

层，充分止血。长条形ADMl cmX4cm大小，以长边中点连线为界，一半打孔，一

半不打孔。以脊柱为分界线，用3- 0手术缝合线将ADM缝合固定于肌肉层上，脊柱

右侧为ADM打孔的部分，左侧为未打孔的部分。再以1号线缝合背部皮肤切口，无

菌纱布覆盖，包扎，适当固定，定期换药，l O天左右拆线。

分别于手术后3天、l 周、2周任选7只SD大鼠，麻醉后切开背部皮肤及皮下