苹果园套栽野生款冬技术

款冬 Tussilago farfara L .为菊科款冬属多年生草本植物 ,以未开放的花蕾入药 ,有止咳化痰 ,润肺下气的功效 ,主产甘肃、河南、四川等地。近年来由于需求的加大 ,导致了毫无节制的乱采乱挖 ,使野生资源大量减少 ,从而使其货紧价扬。野生款冬在半阴半阳的环境和表土疏松、肥沃、湿润的微砂质土壤中生长良好。若在阳光直接照晒下会茎叶萎蔫 ,以至死亡。根据野生品喜阴湿 ,畏日光的特性。试验在盛果期苹果园立体间做的方法 ,利用果树下荫凉、潮湿、含腐殖质丰富的特点 ,在不影响果树生长的情况下 ,试验家种野生款冬 ,现将这一技术报道如下。1…     

胰岛素干预后脂肪干细胞旁分泌对人血管内皮细胞的作用

目的 了解胰岛素刺激后脂肪干细胞(ADSC)旁分泌因子的变化及其对人血管内皮细胞的作用.方法 (1)分离培养人ADSC,按照随机数字表法将细胞分为胰岛素刺激组(含终浓度1×10-7mol/L胰岛素的无血清DMEM培养液培养)和对照组(未加胰岛素的无血清DMEM培养液培养),每组6孔.3 d后收集ADSC培养上清液(ADSC-CM),ELISA法测定血管内皮生长因子(VEGF)、肝细胞生长因子(HGF)表达量.(2)分离培养人脐静脉内皮细胞(HUVEC),将细胞按照随机数字表法分为4组,均采用内皮细胞专用培养液培养:胰岛素刺激ADSC-CM组,培养液中加入经胰岛素刺激后的ADSC-CM;无刺激ADSC-CM组,培养液中含无胰岛素刺激的ADSC-CM;胰岛素组,培养液中加入终浓度为1×10-7mol/L的胰岛素;空白对照组,培养液中不添加刺激因素.3 d后噻唑蓝法测定HUVEC增殖情况,数据用吸光度值表示.(3)另取HUVEC同上分为4组,培养12 h时,膜联蛋白V-异硫氰酸荧光素双染色法测定细胞凋亡情况.(4)另取HUVEC同上分为4组后,体外细胞划痕法测定划痕后12、24、36、48 h时HUVEC迁移距离.对实验数据行t检验.结果 (1)胰岛素刺激组VEGF、HGF含量分别为(643±64)、(930±68)pg/mL,显著高于对照组的(287±47)、(577±84)pg/mL(t值分别为18.869、18.475,P值均小于0.05).(2)胰岛素刺激ADSC-CM组、无刺激ADSC-CM组吸光度值分别为0.847±0.042、0.798±0.022,均高于胰岛素组的0.665±0.028(t值分别为4.579、3.732,P值均小于0.01)及空白对照组的0.674±0.031(t值分别为3.761、4.073,P值均小于0.01);胰岛素刺激ADSC-CM组吸光度值较无刺激ADSC-CM组明显增高(t=2.576,P＜0.05).(3)胰岛素刺激ADSC-CM组、无刺激ADSC-CM组细胞凋亡率分别为(5.8±1.9)%、(9.0±2.0)%,均明显低于胰岛素组的(30.4±6.0)%(t值分别为12.891、10.417,P值均小于0.05)和空白对照组的(31.4±7.4)%(t值分别为11.474、9.783,P值均小于0.05);无刺激ADSC-CM组细胞凋亡率高于胰岛素刺激ADSC-CM组(t=8.548,P＜0.05).(4)划痕后36、48 h时,胰岛素刺激ADSC-CM组、无刺激ADSC-CM组HUVEC迁移距离明显长于胰岛素组与空白对照组,且胰岛素刺激ADSC-CM组细胞迁移距离大于无刺激ADSC-CM组(t值分别为4.076、4.573,P值均小于0.05).结论 胰岛素干预后ADSC旁分泌能更有效促进人血管内皮细胞增殖、迁移并抑制其凋亡,有利于组织血管化.

Abstract：

Objective To study the biological effects of the paracrine from ADSC after being stimulated by insulin on vascular endothelial cells. Methods(1) ADSC was isolated from human adipose tissue and cultured in vitro. The third generation cells were collected and divided into insulin group (1, cultured with serum-free DMEM containing 1 × 10-7 mol/L insulin) and control group (C, cultured with serum-free DMEM) according to the random number table, with 6 slots in each group. Three days later, ADSC culture medium (ADSC-CM) was collected for determination of levels of vascular endothelial growth factor (VEGF) and hepatocyte growth fator (HGF) by ELISA. (2) Human umbilical vein endothelial cells (HUVEC) were cultured to the third generation, and they were cultured with special nutrient solution and divided into ADSC-CM with insulin stimulation group (AI) , ADSC-CM without insulin stimulation group (AC), insulin group(I, with same concentration as above), blank control group (BC) according to the random number table. Three days later, proliferation of HUVEC was determined with MTT method(with expression of absorbance value). Another two samples of HUVEC were respectively divided into 4 groups as above for determination of apoptosis rate with Annexin V/FITC double-staining 12 hours after culture, and H UVEC migration with scratch adhesion test at post scratch hour (PSH) 12, 24, 36, 48. Data were processed with t test.Results(1) Compared with those in C group [(287 ± 47) , (577 ± 84) pg/mL, respectively] , the secretion levels of VEGF and HGF in I group [(643 ±64) , (930 ±68) pg/mL, respectively] were significantly increased(with t value respectively 18. 869, 18. 475, P values all below 0.05). (2) The absorbance value of HUVEC in AI and AC groups was 0. 847 ± 0. 042, 0. 798 ± 0.022, respectively, which were higher than that in I and BC groups [0. 665 ± 0. 028(with t value respectively 4. 579, 3. 732) , 0. 674 ± 0.031(with t value respectively 3. 761, 4. 073) , P values all below 0. 01] , and that in AI group was higher than that in AC group(t =2.576, P ＜0.05). The apoptosis rates of HUVEC in AI and AC groups [(5.8±1.9)%,(9.0 ± 2.0)%, respectively] were obviously lower as compared with that in I and BC groups [(30.4 ±6. 0) %(with t value respectively 12. 891, 10.417),(31.4 ± 7.4) %(with t value respectively 11. 474,9. 783), P values all below 0. 05], and that in AC group was higher than that in AI group(t = 8. 548, P ＜0. 05). The distance of migration of HUVEC in AI and AC groups were greater than that in I and BC groups at PSH 36, 48, and that in AI group was greater as compared with that in AC group(with t value respectively 4.076, 4. 573, P values all below 0. 05). Conclusions Paracrine from ADSC after being stimulated by insulin can promote proliferation and migration of HUVEC, and suppress its apoptosis, and it is beneficial for tissue vascularization.     

人脐静脉血管内皮细胞体外培养的方法研究

目的建立人脐静脉血管内皮细胞(human umbilical vein endothelial cells,HUVECs)体外高效稳定培养的方法,为组织工程及相关医学基础研究提供稳定的细胞来源。方法取自愿捐赠足月妊娠分娩的新生儿脐带,用自制空针软管静脉注入0.1%Ⅱ型胶原酶,置于37℃培养箱中,消化收集,采用含5%FBS及1%内皮细胞生长因子(endothelial cell growth factor,ECGS)的内皮细胞专用培养基进行培养。将消化前后的脐带标本行HE染色,观察HUVECs脱壁情况;流式细胞仪检测原代细胞纯度;细胞培养期间于倒置相差显微镜下观察细胞形态;取第3代HUVECs行免疫细胞化学染色观察、MTT检测细胞增殖情况,并将细胞接种于细胞外基质胶Matrigel上培养24 h,观察细胞管腔形成情况。结果经Ⅱ型胶原酶消化后的脐带内HUVECs大量脱落,细胞消化完全。经Ⅱ型胶原酶37℃培养箱中消化15 min后,可获得纯度为99.56%的HUVECs;原代HUVECs培养后2～3 d生长最快,呈典型的铺路石或鹅卵石样排列,4～6 d融合成片。MTT法检测显示第3代HUVECs培养后3～4 d细胞生长最快,5 d左右融合;免疫细胞化学染色显示内皮细胞Ⅷ因子相关抗原表达阳性,培养24 h后在细胞外基质胶Matrigel上可见类似于毛细血管的完整闭合管腔形成。结论经自制空针软管静脉注入0.1%Ⅱ型胶原酶,使静脉充分充盈,内皮消化完全,采用含5%FBS和1%ECGS的内皮细胞专用培养基,可迅速获取大量高纯度、高存活率的HUVECs。     

人表皮干细胞改良培养及其组织工程皮肤的构建

目的：改良传统人表皮干细胞（hESCs）分离、培养方法,为组织工程皮肤构建提供产率更高、活力更好的种子细胞。方法：应用改良的酶消化法（改良法）及传统酶消化法（传统法）分离、培养hESCs,倒置显微镜观察细胞形态,MTT法绘制生长曲线、免疫组化法测定hESCs标志物角蛋白19（K19）和β1整合素的表达,并且进一步比较上述两种方法所获种子细胞构建的组织工程皮肤的形态学的特性。结果：台盼蓝染色显示改良法细胞消化分离数为92.25±15.61个,传统法细胞消化分离数为68.50±26.91个（P〈0.01）;改良法活细胞比率是（94±0.01）%,传统法活细胞比率是（75±0.04）%（P〈0.05）。在8天时改良法细胞MTT法OD值为1.300,传统法为0.779,改良法较传统法活力好,增殖快;细胞免疫组织化学染色显示表皮干细胞标志物角蛋白19（K19）和β1整合素均为阳性染色;HE染色结果显示采用改良法hESCs构建的组织工程皮肤表皮细胞复层层数为5～6层、基底细胞排列整齐,传统法hESCs构建的组织工程皮肤表皮细胞复层层数为3～4层、基底细胞排列散乱。结论：利用改良的酶消化法可获得数量更多、活力更好的人表皮干细胞,为以hESCs为种子细胞构建组织工程皮肤奠定基础。