基于石蜡切片技术的家蚕胚胎发育与Mago蛋白定位研究
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摘要
探索并建立适合家蚕卵的石蜡切片技术体系；获得家蚕典型胚胎发育时期的显微实体照片，印证补充前人关于家蚕胚胎发育的文字描述、示意图或手绘图，为家蚕胚胎发育基因功能研究提供形态学参考照片；探索并初步建立家蚕免疫组化技术体系，为在组织和细胞水平研究基因功能奠定基础。
以KOH溶液作为卵壳软化液，设置浓度梯度和软化时间梯度，摸索最佳软化时间；摸索适合家蚕卵的固定、透明、染色方法；利用已建立的石蜡切片技术体系，以不同发育时期的家蚕日系N4品种非滞育卵为材料进行石蜡切片，获得较为系统的典型胚胎发育时期的显微实体照片；以家蚕日系N4品种5龄3天幼虫精巢为材料，以家蚕Mago蛋白IgG为Ⅰ抗，摸索免疫组化方法，建立免疫组化技术体系，获得家蚕Mago蛋白组织定位信息。研究结果如下：
1．以家蚕日系N4品种非滞育卵为材料，用10％KOH溶液软化卵壳5min，用Smith液固定，用松油醇(Terpineol)脱水兼透明，建立了家蚕卵石蜡切片技术体系。
2．获得了较为系统的家蚕典型胚胎发育时期的实体照片，这些时期包括胚盘形成、胚带形成、中胚层分化、头胸部突起显现、腹部突起显现、反转前、反转、反转后、器官形成等典型发育时期。还观察到了卵黄细胞的形成过程，区分了合胞体胚盘和细胞胚盘，得到了胚胎中肠、背血管、神经索、生殖腺、丝腺等组织图片。用连续切片技术观察到了类原始生殖细胞存在于中胚层突起处。
3．影响免疫组化结果的因素主要有抗体的纯度，Ⅰ抗和Ⅱ抗最佳稀释度组合，抗原修复、封闭、洗涤、抗体孵育、显色等步骤的时间等。本研究确定的Ⅰ抗和Ⅱ抗最佳稀释度组合为Ⅰ抗1：1000，Ⅱ抗1：500。
4．家蚕Mago蛋白精巢组织的免疫定位结果表明，家蚕Mago蛋白存在于整个精巢组织中，但主要位于精母细胞、精子束细胞和结缔组织细胞的细胞核内。
总之，通过本研究，建立了一套家蚕卵石蜡切片技术体系，具有简便、快速、完整性好、能大批量进行切片等优点，能够满足家蚕胚胎发育基因功能研究的需要；获得了较为系统的家蚕典型胚胎发育时期的实体照片，印证补充了前人关于家蚕胚胎发育的文字描述、示意图、手绘图；探索了家蚕免疫组织化学技术，并对其中的关键环节进行了探讨和标准化，初步建立了一项可重复、稳定性好、适合家蚕组织的蛋白定位方法，获得了家蚕Mago蛋白在家蚕精巢组织的定位结果。
To obtain embryonic microphotographs of representative developmental stages, a paraffin section technical method for silkworm (Bombyx mori) eggs need be explored and established. Then, these microphotographs can conform and enrich the ancestral literal details, schematic diagrams, hand-drawing pictures on this study. In addition, it is believed that these microphotographs may also provide morphology reference for embryonic developmental gene function research in silkworm. To immunolocalize protein in silkworm testis, immunohistochemical staining method must be explored and applied, for the purpose of providing a valuable basis on further gene functional research on tissue and cell level.
To find the best softening time of silkworm eggs using KOH as softening fluid, concentration gradient and softening time gradient have been tested. The method of fixation, clearing and staining have also been tried. Based on the established paraffin section technical method, a lot of embryonic microphotographs were gained from eggs of N4, a Japanese non- diapause strain. The Immunolocalization of BmMago was completed by immunohistochemical(IHC) staining method, which was explored and established with testis on day 3 of fifth larva, using IgG of BmMago as primary antibody. Some results of the study is as follows:
1. Establishment of paraffin section technical method for silkworm (Bombyx mori) eggs. In details, with the eggs of Japanese non- diapause strain named N4, softening with 10% KOH for 5 min, fixing with Smith fixative, de-hydrating and clearing with Terpineol.
2. A lot of embryonic microphotographs from representative developmental stages have been obtained, including stages of blastderm formation, germband formation, mesoderm differentiation, appearance of gnathal and thoracic appendages, appearance of abdominal appendages, early blastokinesis, middle blastokinesis, final blastokinesis, completion of organs and tissues. Moreover, process of yolk cleavage was surveyed; cellular blastoderm and syncytial blastoderm were distinguished; embryonic pictures were captured from different tissue eg. mid-gut, dorsal vessel, nerve cord, gonad, silk gland. The primordial germ-like cells were observed in mesoderm by serial cross section.
3. There were many factors contribute to IHC, such as purity of primary antibody, dilution degree of primary antibody and secondary antibody, time used in the process of retrieval, block, rinse, antibody incubation, coloration. The best dilution degree in this study was 1:1000 of primary antibody and 1:500 of secondary antibody.
4. The Immunolocalization of BmMago revealed that BmMago protein was expressed in all the cells of the silkworm testis, where it was predominantly localized to the nuclei.
In conclusion, a new paraffin section technical method for silkworm {Bombyx mori) eggs has been established, which meet developmental gene functional research, has the advantages of simple, fast, integrity, high throughput. A lot of embryonic microphotographs from representative developmental stages were gained, which conformed and enriched the ancestral literal details, schematic diagrams, hand-drawing pictures on the research field. A immunohistochemical(IHC) staining method has been explored in silkworm, in which key steps have been tested and standardized. BmMago has been immunolocalized in silkworm testis.
以KOH溶液作为卵壳软化液，设置浓度梯度和软化时间梯度，摸索最佳软化时间；摸索适合家蚕卵的固定、透明、染色方法；利用已建立的石蜡切片技术体系，以不同发育时期的家蚕日系N4品种非滞育卵为材料进行石蜡切片，获得较为系统的典型胚胎发育时期的显微实体照片；以家蚕日系N4品种5龄3天幼虫精巢为材料，以家蚕Mago蛋白IgG为Ⅰ抗，摸索免疫组化方法，建立免疫组化技术体系，获得家蚕Mago蛋白组织定位信息。研究结果如下：
1．以家蚕日系N4品种非滞育卵为材料，用10％KOH溶液软化卵壳5min，用Smith液固定，用松油醇(Terpineol)脱水兼透明，建立了家蚕卵石蜡切片技术体系。
2．获得了较为系统的家蚕典型胚胎发育时期的实体照片，这些时期包括胚盘形成、胚带形成、中胚层分化、头胸部突起显现、腹部突起显现、反转前、反转、反转后、器官形成等典型发育时期。还观察到了卵黄细胞的形成过程，区分了合胞体胚盘和细胞胚盘，得到了胚胎中肠、背血管、神经索、生殖腺、丝腺等组织图片。用连续切片技术观察到了类原始生殖细胞存在于中胚层突起处。
3．影响免疫组化结果的因素主要有抗体的纯度，Ⅰ抗和Ⅱ抗最佳稀释度组合，抗原修复、封闭、洗涤、抗体孵育、显色等步骤的时间等。本研究确定的Ⅰ抗和Ⅱ抗最佳稀释度组合为Ⅰ抗1：1000，Ⅱ抗1：500。
4．家蚕Mago蛋白精巢组织的免疫定位结果表明，家蚕Mago蛋白存在于整个精巢组织中，但主要位于精母细胞、精子束细胞和结缔组织细胞的细胞核内。
总之，通过本研究，建立了一套家蚕卵石蜡切片技术体系，具有简便、快速、完整性好、能大批量进行切片等优点，能够满足家蚕胚胎发育基因功能研究的需要；获得了较为系统的家蚕典型胚胎发育时期的实体照片，印证补充了前人关于家蚕胚胎发育的文字描述、示意图、手绘图；探索了家蚕免疫组织化学技术，并对其中的关键环节进行了探讨和标准化，初步建立了一项可重复、稳定性好、适合家蚕组织的蛋白定位方法，获得了家蚕Mago蛋白在家蚕精巢组织的定位结果。
To obtain embryonic microphotographs of representative developmental stages, a paraffin section technical method for silkworm (Bombyx mori) eggs need be explored and established. Then, these microphotographs can conform and enrich the ancestral literal details, schematic diagrams, hand-drawing pictures on this study. In addition, it is believed that these microphotographs may also provide morphology reference for embryonic developmental gene function research in silkworm. To immunolocalize protein in silkworm testis, immunohistochemical staining method must be explored and applied, for the purpose of providing a valuable basis on further gene functional research on tissue and cell level.
To find the best softening time of silkworm eggs using KOH as softening fluid, concentration gradient and softening time gradient have been tested. The method of fixation, clearing and staining have also been tried. Based on the established paraffin section technical method, a lot of embryonic microphotographs were gained from eggs of N4, a Japanese non- diapause strain. The Immunolocalization of BmMago was completed by immunohistochemical(IHC) staining method, which was explored and established with testis on day 3 of fifth larva, using IgG of BmMago as primary antibody. Some results of the study is as follows:
1. Establishment of paraffin section technical method for silkworm (Bombyx mori) eggs. In details, with the eggs of Japanese non- diapause strain named N4, softening with 10% KOH for 5 min, fixing with Smith fixative, de-hydrating and clearing with Terpineol.
2. A lot of embryonic microphotographs from representative developmental stages have been obtained, including stages of blastderm formation, germband formation, mesoderm differentiation, appearance of gnathal and thoracic appendages, appearance of abdominal appendages, early blastokinesis, middle blastokinesis, final blastokinesis, completion of organs and tissues. Moreover, process of yolk cleavage was surveyed; cellular blastoderm and syncytial blastoderm were distinguished; embryonic pictures were captured from different tissue eg. mid-gut, dorsal vessel, nerve cord, gonad, silk gland. The primordial germ-like cells were observed in mesoderm by serial cross section.
3. There were many factors contribute to IHC, such as purity of primary antibody, dilution degree of primary antibody and secondary antibody, time used in the process of retrieval, block, rinse, antibody incubation, coloration. The best dilution degree in this study was 1:1000 of primary antibody and 1:500 of secondary antibody.
4. The Immunolocalization of BmMago revealed that BmMago protein was expressed in all the cells of the silkworm testis, where it was predominantly localized to the nuclei.
In conclusion, a new paraffin section technical method for silkworm {Bombyx mori) eggs has been established, which meet developmental gene functional research, has the advantages of simple, fast, integrity, high throughput. A lot of embryonic microphotographs from representative developmental stages were gained, which conformed and enriched the ancestral literal details, schematic diagrams, hand-drawing pictures on the research field. A immunohistochemical(IHC) staining method has been explored in silkworm, in which key steps have been tested and standardized. BmMago has been immunolocalized in silkworm testis.
引文
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