| Objective: With the development and improvement of the society ,our living standard has been raised unceasingly and life mode has been changed,but attack rate of diabetes has gradually upgraded in our country and in the world . Diabetes can cause complications on eye,kidney,nerves,blood vessel and so on.By time at present,there are a deal of reports about the research of diabetic retinopathy and diabetic nephropathy in exterior and interior.but about diabetic skeletal muscle process,there is less research.Skeletal muscle is one of the capital tissues about utilizing glucose in all over the body,also the main target site that hyperglycemia impaired.Both hyperglycemia and low limb ischemia can cause low limb skeletal's pathological change.In recent years,the effect of growth factor has been thunk highly of in the growth and development of skeletal muscle ,vascularization of muscle and repair following injury.With the advancement of stem cell's research and improvement of separating and purifying stem cell,it is possible to re-establish effective collateral branch circulation in ischemic position by stem cell therapy.At present , in clinic it is been confirmed that autologoous peripheral blood stem cell transplantation(ABSCT) is a fine method of curing diabetic low limb ischemia.This research makes a copy of rat model of T1DM by intraperitoneal injection STZ in SD rats,then we deligate arteria cruralis and mutilate it to manufacture diabetic lower limb ischemia model.We enforce ABSCT in part rats ,we observe ABSCT impact the expression of blood vessel endothelium growth factor (VEGF) and basic fibroblast growth factor(bFGF) in low limb's gastrocnemius ,we approach that ABSCT improve diabetic skeletal muscle's pathological changes in rats .Thereby we will provid theory evidence about curing diabetic skeletal muscle's pathological change for enforcing stem cell transplant in clinic.Methods:1 The preparation of diabetic modelthere are 45 heathy SD masculinity rats, then they will be divided into 3 groups in random after being fed for one week:normal control (A group) ,diabetic lower limb ischemia(B group) and stem cell transplant after diabetic lower limb ischemia(C group),each group has 15 rats.We will copy T1DM model by intraperitoneal injection STZ 65mg/kg in B group and C group after 12 hours on an empty stomach ,we will measure blood glucose after 72 hours of injecting STZ,if blood glucose exceeds 16.65 mmol/L , we think models are done well.2 The preparation of diabetic lower limb ischemia'modelWe will deligate both lower extremities'femoral artery and mutilate it to make models of diabetic lower limb ischemia after 7 days of diabetic model done well. then we will prevent wound infection by intramuscular injection of 4 pan laevomycetin in each rat for 3 days .there are 2 death in B group ,there is 1 death in C group.3 mobilization of stem cellIn the 7th day of ischemia model done well , We will administrate rhG-CSF of 15μg.kg-1.D-1, we use it for 3 days,we will collect venous blood to monitoring white blood cell(WBC) in the third day.4 The enforcing of ABSCTIn the fourth day,we will get 5 ml blood from the heart to separate mononuclearcell in each rat,then we will inject mononuclearcell into gastrocnemius of left lower extremity .5 Specimen collectionwe will get blood from heart after 4 weeks of stem cell transplant ,then we put rats to death and remain gastrocnemius of lower limb in 10% formalin.we can observe pathological consequence of three groups by HE staining and measure the expression of VEGF and bFGF in gastrocnemius of lower limb by immunohistochemistry.6 Observing index6.1 Blood glucose is measured by glucose oxidase methodProcedure of determination:preparing 3 tubes:blank tube,standard tube,determining tube,we put 1.5 ml enzyme and 1.5 ml phenol regent,we put standard liquid into standard tube,we put sample into determining tube,after misce bene we keep in 37℃for 15 minutes,we make zero setting with blank tube and measure absorbance of each tube.determining parameter:wave length 505nm,temperature 37℃.6.2 The determination of white blood cell getting 1 ml blood ,counting WBC utilizing FCM6.3 The examination of gastrocnemius histomorphologyPreparing the light microscope of rat's gastrocnemius,making HE staining and test under microscope.6.4 The expression of VEGF and bFGF in gastrocnemiusDetermining the expression of VEGF and bFGF in gastrocnemius by immunohistochemistry .6.5 test under microscope Observing skeletal muscle morphology under lightmicroscope, observing the epression of VEGF and bFGF, endochylema is put color in VEGF and bFGF, masculine cell is buffy,with PBS as negative control,we define non-expression as (-),low expression as (+),midrange expression as (++),high expression as (+++),we choose 50 leaf cut sheet to observe under high power lens.7 Statistical treatmentAll data will be dealed with SPSS 13.0 , measurement data will be tested with t test of mean,above two sets adopt analysis of variance of mono-factor, the compare of expression of VEGF and bFGF use non-parametric test,docimastic significance is expressed with P.if P<0.05,difference is conspicuous, if P<0.01,difference is very conspicuous.Results:1 there are evident polydipsia, polyphagia, hyperdiuresis, sequential body weight is manifest decreasing .B group rats have pale in tip of doigt and shin temperature is decreasing.after 4 weeks of cellular transplant,pale is relief in left hind limb compared with right hind limb,skin temperature is getting up,skin temperature of left hind limb in C group has none manifest decreasing compared with A group.2 blood glucose : after DM model done well,blood glucose of B group is obviously higher than A group,they are significant difference(P<0.05), blood glucose of C group is obviously higher than A group,they are significant difference(P<0.05), blood glucose of B group have no significant difference compared with C group(P>0.05).3 measurement of WBCAfter 3 days of mobilizing stem cell, WBC of C group is higher than Agroup,there is significant difference(P<0.05), WBC of C group is higher than B group,there is significant difference(P<0.05), WBC of A group have no significant difference compared with B group.4 histologic observation display that skeletal muscle of normal control is normal tissue. About stem cell transplant group after diabetic low limb ischemia it is thus evident that endochylema kytoplasm of skeletal muscle is to condense, cell nucleus is pyknosis,part area of muscle fiber is breaking.About diabetic low limb ischemia group skeletal muscle is general atrophy,myofibrosis and necrosis, cell nucleus is pyknosis,transverse is ambiguity and disappear , interstitial fatty tissue is hyperplasia.5 The expression of VEGF and bFGF in gastrocnemiusIn the skeletal muscle of A group , there is expression of bFGF, positive cell is buffy,the expression of bFGF in B group and C group is attenuated, they have statistical significance(P<0.05) .the expression of bFGF in left hind limb is upgraded compared with B group,the compare of 2 groups has statistical significance (P<0.05).There is no expression of VEGF in skeletal muscle of A group, There is expression of VEGF in skeletal muscle of B Group and C group, the expression of VEGF in B group is lower than C group, the compare of 2 group has statistical significance (P<0.05).Conclusions:1 intraperitoneal injection of STZ and deligating femoral artery of lower limb can manufacture the model of rat's diabetic skeletal muscle pathological changes.2 Normal skeletal muscles do not express VEGF,ischemia can make expression of VEGF in skeletal muscle,ABSCT can make higher expression of VEGF ,thereby ABSCT can release lower limb ischemia of diabetes,and release the progress of diabetic skeletal muscle.3 there is expression of bFGF in normal skeletal muscle, ischemia can make low expression of bFGF in skeletal muscle , ABSCT can remittence lower expression of bFGF, thereby it can raise blood vessel neogenesis of lower limb and step down the progress of diabetic skeletal muscle.
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