The aseptic seedlings of Albizia julibrissin were divided into two groups. One group was inoculated by rhizobia and the other was designed as control without inoculation. These two groups were all cultivated in steril...The aseptic seedlings of Albizia julibrissin were divided into two groups. One group was inoculated by rhizobia and the other was designed as control without inoculation. These two groups were all cultivated in sterilized cultured liquid for about two weeks. In the first group,the root hairs did not form on the main roots,but formed a lot on the base part of the lateral roots. In the second group, the root hairs did not form not only on the main roots but also on the lateral roots. The root segments which have distinctive deformation of root hairs from first group were cut off and fixed, in the meanwhile the corresponding root segments were cut off from the contrastive group and fixed. These two groups of roots segments were treated as semi-thin section and ultrathin sections, then were observed by optical microscope and transmission electron microscope. The result showed that the transfer cells with wall ingrowths on the tangential exowall of the root’s epiderm and exodermis cells, are easy to be found in the root segments which has distinctive deformation of root hair. The wall ingrowth lies not only in root epidermal cells but also in the root exodermis cells, while there is no wall ingrowth in root epiderm and exodermis cells of A. julibrissin which is not inoculated by rhizobia. This suggested that the forming of root hairs and transfer cells in epiderm and exodermis of A. julibrissin is resulted from the inducement of rhizobia of A. julibrissin.展开更多
目的研究新霉素对大鼠前庭毛细胞的毒性作用并探索前庭基因导入的方法和途径,为药物性前庭功能损伤动物模型的制备和基因治疗的相关研究提供参考。方法将25只成年Wistar大鼠分为正常对照组、新霉素鼓阶给药组、新霉素前庭阶给药组、腺病...目的研究新霉素对大鼠前庭毛细胞的毒性作用并探索前庭基因导入的方法和途径,为药物性前庭功能损伤动物模型的制备和基因治疗的相关研究提供参考。方法将25只成年Wistar大鼠分为正常对照组、新霉素鼓阶给药组、新霉素前庭阶给药组、腺病毒(缺失E1、E3基因且构建有绿色荧光蛋白报告基因的复制缺陷型腺病毒,Ad-EGFP)鼓阶导入组、腺病毒(Ad-EGFP)前庭阶导入组,每组各5只动物。新霉素组大鼠在右耳通过耳蜗底回鼓阶(鼓阶组)或前庭阶打孔(前庭组)的方法在耳蜗内注入0.1%新霉素5μl,Ad-EGFP组则以同法导入物理滴度为2.1×1011vp/ml的Ad-EGFP 5μl,正常对照组大鼠不做任何处理。处理3天后对动物进行颈髓硬膜外短声诱发电位(click-evoked potentials on the surface of the cervical dura mater,CDM-CEP)检查和游泳试验,评价前庭功能,然后将动物处死,进行组织学和形态学观察。结果3天后新霉素组大鼠的前庭毛细胞即出现严重破坏,并出现严重的前庭功能损伤。正常对照组、新霉素鼓阶给药组、新霉素前庭阶给药组、Ad-EGFP鼓阶导入组、Ad-EGFP前庭阶导入组的游泳时间分别为4.0±0.71、10.2±1.64、9.8±1.48、4.8±0.84、5.0±0.71 s;正常对照组CDM-CEP的阈值为85±3.54 dB SPL,潜伏期为6.59±0.41 s;新霉素组120 dB SPL未引出CDM-CEP;Ad-EGFP鼓阶导入组CDM-CEP阈值为91±5.48 dB SPL,潜伏期为6.76±0.26 s,Ad-EGFP新霉素前庭阶导入组CDM-CEP的阈值为89±6.52 dB SPL,潜伏期为6.78±0.26 s。3天后,Ad-EGFP前庭阶导入组的前庭终末器官均出现Ad-EGFP的转染;而Ad-EGFP鼓阶导入组未见前庭终末器官的表达。结论新霉素对前庭毛细胞具有极强的破坏作用,新霉素耳蜗内注射的方法可以制备理想的前庭功能障碍动物模型,耳蜗底回前庭阶打孔可以作为基因导入前庭的有效途径。展开更多
In the present study, cashmere goat fetal fibroblasts were transfected with pCDsR-KI, a hair-follicle-cell specific expression vector for insulin-like growth factor 1 (IGF1) that contains two markers for selection (re...In the present study, cashmere goat fetal fibroblasts were transfected with pCDsR-KI, a hair-follicle-cell specific expression vector for insulin-like growth factor 1 (IGF1) that contains two markers for selection (red fluorescent protein gene and neomycin resistant gene). The transgenic fibroblasts cell lines were obtained after G418 selection. Prior to the somatic cell nuclear transfer (SCNT), the maturation rate of caprine cumulus oocytes complexes (COCs) was optimized to an in vitro maturation time of 18 h. Parthenogenetic ooctyes were used as a model to investigate the effect of two activation methods, one with calcium ionophore IA23187 plus 6-DMAP and the other with ethanol plus 6-DMAP. The cleavage rates after 48 h were respectively 88.7% and 86.4%, with no significant difference (P>0.05). There was no significant difference between the cleavage rate and the blastocyst rate in two different media (SO- Faa and CR1aa; 86.3% vs 83.9%, P>0.05 and 23.1% vs 17.2%,P>0.05). The fusion rate of a 190 V/mm group (62.4%) was significantly higher than 130 V/mm (32.8%) and 200 V/mm (42.9%), groups (P<0.05). After transgenic somatic cell nuclear transfer (TSCNT) manipulation, 203 reconstructed embryos were obtained in which the cleavage rate after in vitro development (IVD) for 48 h was 79.3% (161/203). The blastocyst rate after IVD for 7 to 9 d was 15.3% (31/203). There were 17 embryos out of 31 strongly ex- pressing red fluorescence. Two of the red fluorescent blastocysts were randomly selected to identify transgene by polymerase chain reaction. Both were positive. These results showed that: (i) RFP and Neor genes were correctly expressed indicating that transgenic somatic cell lines and positive trans- genic embryos were obtained; (ii) one more selection at the blastocyst stage was necessary although the donor cells were transgenic positive, because only partially transgenic embryos expressing red fluorescence were obtained; and (iii) through TSCNT manipulation and optimization, transgenic cash- mere goat embryos expressing red fluorescence and containing an IGF1 expression cassette were obtained, which was sufficient for production of transgenic cashmere goats.展开更多
文摘The aseptic seedlings of Albizia julibrissin were divided into two groups. One group was inoculated by rhizobia and the other was designed as control without inoculation. These two groups were all cultivated in sterilized cultured liquid for about two weeks. In the first group,the root hairs did not form on the main roots,but formed a lot on the base part of the lateral roots. In the second group, the root hairs did not form not only on the main roots but also on the lateral roots. The root segments which have distinctive deformation of root hairs from first group were cut off and fixed, in the meanwhile the corresponding root segments were cut off from the contrastive group and fixed. These two groups of roots segments were treated as semi-thin section and ultrathin sections, then were observed by optical microscope and transmission electron microscope. The result showed that the transfer cells with wall ingrowths on the tangential exowall of the root’s epiderm and exodermis cells, are easy to be found in the root segments which has distinctive deformation of root hair. The wall ingrowth lies not only in root epidermal cells but also in the root exodermis cells, while there is no wall ingrowth in root epiderm and exodermis cells of A. julibrissin which is not inoculated by rhizobia. This suggested that the forming of root hairs and transfer cells in epiderm and exodermis of A. julibrissin is resulted from the inducement of rhizobia of A. julibrissin.
文摘目的研究新霉素对大鼠前庭毛细胞的毒性作用并探索前庭基因导入的方法和途径,为药物性前庭功能损伤动物模型的制备和基因治疗的相关研究提供参考。方法将25只成年Wistar大鼠分为正常对照组、新霉素鼓阶给药组、新霉素前庭阶给药组、腺病毒(缺失E1、E3基因且构建有绿色荧光蛋白报告基因的复制缺陷型腺病毒,Ad-EGFP)鼓阶导入组、腺病毒(Ad-EGFP)前庭阶导入组,每组各5只动物。新霉素组大鼠在右耳通过耳蜗底回鼓阶(鼓阶组)或前庭阶打孔(前庭组)的方法在耳蜗内注入0.1%新霉素5μl,Ad-EGFP组则以同法导入物理滴度为2.1×1011vp/ml的Ad-EGFP 5μl,正常对照组大鼠不做任何处理。处理3天后对动物进行颈髓硬膜外短声诱发电位(click-evoked potentials on the surface of the cervical dura mater,CDM-CEP)检查和游泳试验,评价前庭功能,然后将动物处死,进行组织学和形态学观察。结果3天后新霉素组大鼠的前庭毛细胞即出现严重破坏,并出现严重的前庭功能损伤。正常对照组、新霉素鼓阶给药组、新霉素前庭阶给药组、Ad-EGFP鼓阶导入组、Ad-EGFP前庭阶导入组的游泳时间分别为4.0±0.71、10.2±1.64、9.8±1.48、4.8±0.84、5.0±0.71 s;正常对照组CDM-CEP的阈值为85±3.54 dB SPL,潜伏期为6.59±0.41 s;新霉素组120 dB SPL未引出CDM-CEP;Ad-EGFP鼓阶导入组CDM-CEP阈值为91±5.48 dB SPL,潜伏期为6.76±0.26 s,Ad-EGFP新霉素前庭阶导入组CDM-CEP的阈值为89±6.52 dB SPL,潜伏期为6.78±0.26 s。3天后,Ad-EGFP前庭阶导入组的前庭终末器官均出现Ad-EGFP的转染;而Ad-EGFP鼓阶导入组未见前庭终末器官的表达。结论新霉素对前庭毛细胞具有极强的破坏作用,新霉素耳蜗内注射的方法可以制备理想的前庭功能障碍动物模型,耳蜗底回前庭阶打孔可以作为基因导入前庭的有效途径。
基金Supported by the National High Technology Research and Development Program of China (Grant No. 2002AA242061) Natural Science Foundation of Inner Mongo-lia (Grant No. 200607010405)
文摘In the present study, cashmere goat fetal fibroblasts were transfected with pCDsR-KI, a hair-follicle-cell specific expression vector for insulin-like growth factor 1 (IGF1) that contains two markers for selection (red fluorescent protein gene and neomycin resistant gene). The transgenic fibroblasts cell lines were obtained after G418 selection. Prior to the somatic cell nuclear transfer (SCNT), the maturation rate of caprine cumulus oocytes complexes (COCs) was optimized to an in vitro maturation time of 18 h. Parthenogenetic ooctyes were used as a model to investigate the effect of two activation methods, one with calcium ionophore IA23187 plus 6-DMAP and the other with ethanol plus 6-DMAP. The cleavage rates after 48 h were respectively 88.7% and 86.4%, with no significant difference (P>0.05). There was no significant difference between the cleavage rate and the blastocyst rate in two different media (SO- Faa and CR1aa; 86.3% vs 83.9%, P>0.05 and 23.1% vs 17.2%,P>0.05). The fusion rate of a 190 V/mm group (62.4%) was significantly higher than 130 V/mm (32.8%) and 200 V/mm (42.9%), groups (P<0.05). After transgenic somatic cell nuclear transfer (TSCNT) manipulation, 203 reconstructed embryos were obtained in which the cleavage rate after in vitro development (IVD) for 48 h was 79.3% (161/203). The blastocyst rate after IVD for 7 to 9 d was 15.3% (31/203). There were 17 embryos out of 31 strongly ex- pressing red fluorescence. Two of the red fluorescent blastocysts were randomly selected to identify transgene by polymerase chain reaction. Both were positive. These results showed that: (i) RFP and Neor genes were correctly expressed indicating that transgenic somatic cell lines and positive trans- genic embryos were obtained; (ii) one more selection at the blastocyst stage was necessary although the donor cells were transgenic positive, because only partially transgenic embryos expressing red fluorescence were obtained; and (iii) through TSCNT manipulation and optimization, transgenic cash- mere goat embryos expressing red fluorescence and containing an IGF1 expression cassette were obtained, which was sufficient for production of transgenic cashmere goats.
