目的研究腺病毒携带Math1-EGFP基因经完整圆窗膜途径及鼓阶打孔途径导入耳蜗后对听功能和转导效率的影响,为内耳基因治疗提供实验基础和理论依据。方法健康成年白色红目豚鼠40只,雌雄不限,体重250～300g。随机分成四组,完整圆窗膜组12只...目的研究腺病毒携带Math1-EGFP基因经完整圆窗膜途径及鼓阶打孔途径导入耳蜗后对听功能和转导效率的影响,为内耳基因治疗提供实验基础和理论依据。方法健康成年白色红目豚鼠40只,雌雄不限,体重250～300g。随机分成四组,完整圆窗膜组12只,鼓阶打孔组12只,各组分别设对照8只。实验组(24只)导入重组腺病毒携带的Math1基因及增强型绿色荧光蛋白基因(enhanced green fluorescent protein,EGFP),对照组(16只)导入人工外淋巴液,所有动物均以左耳作为导入耳。术前及术后分别行听性脑干反应(ABR)检查。分别于术后5天、14天取双侧耳蜗标本做基底膜铺片观察基因表达情况。结果完整圆窗膜组导入耳ABR阈值,术后5天各频率与术前比较无显著性差异(P>0.05);鼓阶打孔组导入耳ABR阈值,术后5天在2kHz、4kHz与术前比较无差异(P>0.05),8kHz较术前增高(P<0.05),16kHz、20kHz较术前明显增高(P<0.01),术后14天在16kHz、20kHz较术后5天时明显好转(P<0.01),但较术前仍有增高(P<0.05)。转导成功率鼓阶打孔组为91.6%,优于完整圆窗膜组的50%。两种转导途径对目的基因在耳蜗内的表达部位和表达时间没有显著影响。结论完整圆窗膜途径及鼓阶打孔途径在转导成功率及听功能保护方面各有优劣。完整圆窗膜途径因其对耳蜗的损伤极小,在临床应用方面具有更好的发展前景。展开更多
目的研究以复制缺陷型腺病毒为载体的Math1基因内耳应用的安全性。方法将10只成年Wistar大鼠分为正常对照组和缺失E1、E3基因片段且构建有Math1基因和绿色荧光蛋白报告基因的复制缺陷型腺病毒(adenovirus-Math1-enhanced green fluoresc...目的研究以复制缺陷型腺病毒为载体的Math1基因内耳应用的安全性。方法将10只成年Wistar大鼠分为正常对照组和缺失E1、E3基因片段且构建有Math1基因和绿色荧光蛋白报告基因的复制缺陷型腺病毒(adenovirus-Math1-enhanced green fluorescence protein,Ad-Math1-EGFP)前庭阶导入组(实验组),每组5只,实验组大鼠在右耳通过耳蜗底回前庭阶打孔的方法导入物理滴度为2.1×1011v.p/ml的上述腺病毒5μl,对照组大鼠不做任何处理。7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)、听性脑干反应(ABR)阈值检测和游泳试验,评价前庭和耳蜗功能,然后将动物处死进行组织形态学观察。结果Ad-Math1-EGFP导入7天后,实验组大鼠前庭及耳蜗的毛细胞及纤毛均未见破坏,形态正常。短声刺激下,对照组大鼠CDM-CEP的阈值为85±3.54dBSPL,ABR阈值为37±4.47dBSPL;实验组大鼠CDM-CEP的阈值为89±6.52dBSPL,ABR阈值为40±3.54dBSPL;对照组大鼠的游泳时间为4.0±0.71s,实验组为5.0±0.71s,两组之间比较差异均无统计学意义。结论携带Math1基因的复制缺陷型腺病毒对前庭和耳蜗毛细胞是安全的,可以作为基因导入的理想载体。展开更多
目的比较腺病毒、脂质体、纳米材料三种不同类型的载体体外携带目的基因Math1对HEK293T细胞的转染效率及细胞毒性的大小,以期筛选理想的基因转染载体材料。方法选用重组腺病毒、LipofectamineTM2000、Superfect纳米材料作为转染载体,携...目的比较腺病毒、脂质体、纳米材料三种不同类型的载体体外携带目的基因Math1对HEK293T细胞的转染效率及细胞毒性的大小,以期筛选理想的基因转染载体材料。方法选用重组腺病毒、LipofectamineTM2000、Superfect纳米材料作为转染载体,携带含有增强型绿色荧光蛋白(enhanced green fluorescent protein,EGFP)报告基因的Math1-EGFP基因及真核表达质粒pRK5-Math1-EGFP,并且根据不同转染载体说明步骤优化体外转染条件,分别转染293T细胞。在一定的时间内利用荧光显微镜观察细胞转染结果并计数阳性细胞,采用MTT(噻唑蓝)法检测三种不同载体体外细胞毒性,应用RT-PCR(逆转录聚合酶链反应)技术检测转染阳性细胞中目的基因Math1的mRNA表达情况。结果在优化的体外转染条件下,重组腺病毒载体介导的细胞转染率达到了94%以上,脂质体和商品化Superfect纳米材料转染的细胞中,荧光蛋白表达率分别为73%和80%以上,同时,商品化Superfect纳米材料在达到80%转染率的条件下,细胞存活率为90%。应用RT-PCR方法证实,三种载体转染细胞均有Math1基因的mRNA的表达。结论商品化Superfect纳米材料作为一种新型的、安全有效的纳米转染材料,能够成功携带耳聋治疗关键基因Math1转染293T细胞,以期在内耳基因治疗的研究当中得到有效的应用。展开更多
Hair cell regeneration is the fundamental method of correcting hearing loss and balance disorders caused by hair cell damage or loss. How to promote hair cell regeneration is a hot focus in current research. In mammal...Hair cell regeneration is the fundamental method of correcting hearing loss and balance disorders caused by hair cell damage or loss. How to promote hair cell regeneration is a hot focus in current research. In mammals, cochlear hair cells cannot be regenerated and few vestibular hair cells can be renewed through spontaneous regeneration. However, Math1 gene transfer allows a few inner ear cells to be transformed into hair cells in vitro or in vivo. Hair cells can be renewed through two possible means in birds： supporting cell differentiation and transdifferentiation with or without cell division. Hair cell regeneration is strongly associated with cell proliferation. Therefore, this study explored the relationship between Math1-induced vestibular hair cell regeneration and cell division in mammals. The mouse vestibule was isolated to harvest vestibular epithelial cells. Ad-Math1-enhanced green fluorescent protein （EGFP） was used to track cell division during hair cell transformation.5-Bromo-2′-deoxyuridine （BrdU） was added to track cell proliferation at various time points. Immunocytochemistry was utilized to determine cell differentiation and proliferation. Results demonstrated that when epithelial cells were in a higher proliferative stage, more of these cells differentiated into hair cells by Math1 gene transfer. However, in the low proliferation stage, no BrdU-positive cells were seen after Math1 gene transfer. Cell division always occurred before Math1 transfection but not during or after Math1 transfection, when cells were labeled with BrdU before and after Ad-Math1-EGFP transfection. These results confirm that vestibular epithelial cells with high proliferative potential can differentiate into new hair cells by Math1 gene transfer, but this process is independent of cell proliferation.展开更多
The process of turning forest area into land is known as deforestation or forest degradation. Reforestation as a fraction of deforestation is extremely low. For improved qualitative and quantitative classification, we...The process of turning forest area into land is known as deforestation or forest degradation. Reforestation as a fraction of deforestation is extremely low. For improved qualitative and quantitative classification, we used Sentinel-1 dataset of State of Para, Brazil to precisely and closely monitor deforestation between June 2019 and June 2023. This research aimed to find out suitable model for classification called Satellite Imaging analysis by Transpose deep neural transformation network (SIT-net) using mathematical model based on Band math approach to classify deforestation applying transpose deep neural network. The main advantage of proposed model is easy to handle SAR images. The study concludes that SAR satellite gives high-resolution images to improve deforestation monitoring and proposed model takes less computational time compared to other techniques.展开更多
Malignant neoplasms of the appendix are rare and represent less than 1% of gastrointestinal cancers.Goblet cell carcinoids(GCC) tumors are a distinctive group of heterogeneous appendiceal neoplasm that exhibit unique ...Malignant neoplasms of the appendix are rare and represent less than 1% of gastrointestinal cancers.Goblet cell carcinoids(GCC) tumors are a distinctive group of heterogeneous appendiceal neoplasm that exhibit unique clinical and pathologic features.This review focuses on the current diagnostic procedures,pathogenesis,possible signaling mechanisms and treatment options for GCC.Perspectives for future research are discussed.The tumor likely arises from pluripotent intestinal epithelial crypt base stem cells.Previous findings of Notch signaling as a tumor suppressor in Neuroendocrine tumors may have a similar role in this tumor too.Loss of Notch signaling may be the driver mutation with other successive downstream mutations likely favors them into progressing and behavior similar to poorly differentiated adenocarcinoma with minimal neuroendocrine differentiation.A multidisciplinary approach is suggested for optimal outcomes.Surgery remains the main treatment modality.Simple appendectomy may be sufficient in early stages while right hemicolectomy is recommended for advanced tumors.Cytoreductive surgery with heated intraperitoneal chemotherapy may improve survival in a select few with metastatic peritoneal disease.These tumors have an unpredictable behavior even in early stages and local recurrence and delayed metastases may be seen.Lifelong surveillance is warranted.展开更多
文摘目的研究腺病毒携带Math1-EGFP基因经完整圆窗膜途径及鼓阶打孔途径导入耳蜗后对听功能和转导效率的影响,为内耳基因治疗提供实验基础和理论依据。方法健康成年白色红目豚鼠40只,雌雄不限,体重250～300g。随机分成四组,完整圆窗膜组12只,鼓阶打孔组12只,各组分别设对照8只。实验组(24只)导入重组腺病毒携带的Math1基因及增强型绿色荧光蛋白基因(enhanced green fluorescent protein,EGFP),对照组(16只)导入人工外淋巴液,所有动物均以左耳作为导入耳。术前及术后分别行听性脑干反应(ABR)检查。分别于术后5天、14天取双侧耳蜗标本做基底膜铺片观察基因表达情况。结果完整圆窗膜组导入耳ABR阈值,术后5天各频率与术前比较无显著性差异(P>0.05);鼓阶打孔组导入耳ABR阈值,术后5天在2kHz、4kHz与术前比较无差异(P>0.05),8kHz较术前增高(P<0.05),16kHz、20kHz较术前明显增高(P<0.01),术后14天在16kHz、20kHz较术后5天时明显好转(P<0.01),但较术前仍有增高(P<0.05)。转导成功率鼓阶打孔组为91.6%,优于完整圆窗膜组的50%。两种转导途径对目的基因在耳蜗内的表达部位和表达时间没有显著影响。结论完整圆窗膜途径及鼓阶打孔途径在转导成功率及听功能保护方面各有优劣。完整圆窗膜途径因其对耳蜗的损伤极小,在临床应用方面具有更好的发展前景。
文摘目的研究以复制缺陷型腺病毒为载体的Math1基因内耳应用的安全性。方法将10只成年Wistar大鼠分为正常对照组和缺失E1、E3基因片段且构建有Math1基因和绿色荧光蛋白报告基因的复制缺陷型腺病毒(adenovirus-Math1-enhanced green fluorescence protein,Ad-Math1-EGFP)前庭阶导入组(实验组),每组5只,实验组大鼠在右耳通过耳蜗底回前庭阶打孔的方法导入物理滴度为2.1×1011v.p/ml的上述腺病毒5μl,对照组大鼠不做任何处理。7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)、听性脑干反应(ABR)阈值检测和游泳试验,评价前庭和耳蜗功能,然后将动物处死进行组织形态学观察。结果Ad-Math1-EGFP导入7天后,实验组大鼠前庭及耳蜗的毛细胞及纤毛均未见破坏,形态正常。短声刺激下,对照组大鼠CDM-CEP的阈值为85±3.54dBSPL,ABR阈值为37±4.47dBSPL;实验组大鼠CDM-CEP的阈值为89±6.52dBSPL,ABR阈值为40±3.54dBSPL;对照组大鼠的游泳时间为4.0±0.71s,实验组为5.0±0.71s,两组之间比较差异均无统计学意义。结论携带Math1基因的复制缺陷型腺病毒对前庭和耳蜗毛细胞是安全的,可以作为基因导入的理想载体。
文摘目的比较腺病毒、脂质体、纳米材料三种不同类型的载体体外携带目的基因Math1对HEK293T细胞的转染效率及细胞毒性的大小,以期筛选理想的基因转染载体材料。方法选用重组腺病毒、LipofectamineTM2000、Superfect纳米材料作为转染载体,携带含有增强型绿色荧光蛋白(enhanced green fluorescent protein,EGFP)报告基因的Math1-EGFP基因及真核表达质粒pRK5-Math1-EGFP,并且根据不同转染载体说明步骤优化体外转染条件,分别转染293T细胞。在一定的时间内利用荧光显微镜观察细胞转染结果并计数阳性细胞,采用MTT(噻唑蓝)法检测三种不同载体体外细胞毒性,应用RT-PCR(逆转录聚合酶链反应)技术检测转染阳性细胞中目的基因Math1的mRNA表达情况。结果在优化的体外转染条件下,重组腺病毒载体介导的细胞转染率达到了94%以上,脂质体和商品化Superfect纳米材料转染的细胞中,荧光蛋白表达率分别为73%和80%以上,同时,商品化Superfect纳米材料在达到80%转染率的条件下,细胞存活率为90%。应用RT-PCR方法证实,三种载体转染细胞均有Math1基因的mRNA的表达。结论商品化Superfect纳米材料作为一种新型的、安全有效的纳米转染材料,能够成功携带耳聋治疗关键基因Math1转染293T细胞,以期在内耳基因治疗的研究当中得到有效的应用。
基金supported by the National Natural Science Foundation of China(NSFC)grant No.81420108010,81271084 to FLC,81370022,81570920,81000413 to DR,81200740 to JMY,81200738 to NC,81371093 to ZH,81400460 to ZG,81200739 to JW+2 种基金973 Program,grant No.2011CB504500 and 2011CB504506The Innovation Project of Shanghai Municipal Science and Technology Commission,grant No.11411952300 to FLCthe Training Program of the Excellent Young Talents of the Shanghai Municipal Health System,grant No.XYQ2013084 to DR
文摘Hair cell regeneration is the fundamental method of correcting hearing loss and balance disorders caused by hair cell damage or loss. How to promote hair cell regeneration is a hot focus in current research. In mammals, cochlear hair cells cannot be regenerated and few vestibular hair cells can be renewed through spontaneous regeneration. However, Math1 gene transfer allows a few inner ear cells to be transformed into hair cells in vitro or in vivo. Hair cells can be renewed through two possible means in birds： supporting cell differentiation and transdifferentiation with or without cell division. Hair cell regeneration is strongly associated with cell proliferation. Therefore, this study explored the relationship between Math1-induced vestibular hair cell regeneration and cell division in mammals. The mouse vestibule was isolated to harvest vestibular epithelial cells. Ad-Math1-enhanced green fluorescent protein （EGFP） was used to track cell division during hair cell transformation.5-Bromo-2′-deoxyuridine （BrdU） was added to track cell proliferation at various time points. Immunocytochemistry was utilized to determine cell differentiation and proliferation. Results demonstrated that when epithelial cells were in a higher proliferative stage, more of these cells differentiated into hair cells by Math1 gene transfer. However, in the low proliferation stage, no BrdU-positive cells were seen after Math1 gene transfer. Cell division always occurred before Math1 transfection but not during or after Math1 transfection, when cells were labeled with BrdU before and after Ad-Math1-EGFP transfection. These results confirm that vestibular epithelial cells with high proliferative potential can differentiate into new hair cells by Math1 gene transfer, but this process is independent of cell proliferation.
文摘The process of turning forest area into land is known as deforestation or forest degradation. Reforestation as a fraction of deforestation is extremely low. For improved qualitative and quantitative classification, we used Sentinel-1 dataset of State of Para, Brazil to precisely and closely monitor deforestation between June 2019 and June 2023. This research aimed to find out suitable model for classification called Satellite Imaging analysis by Transpose deep neural transformation network (SIT-net) using mathematical model based on Band math approach to classify deforestation applying transpose deep neural network. The main advantage of proposed model is easy to handle SAR images. The study concludes that SAR satellite gives high-resolution images to improve deforestation monitoring and proposed model takes less computational time compared to other techniques.
文摘Malignant neoplasms of the appendix are rare and represent less than 1% of gastrointestinal cancers.Goblet cell carcinoids(GCC) tumors are a distinctive group of heterogeneous appendiceal neoplasm that exhibit unique clinical and pathologic features.This review focuses on the current diagnostic procedures,pathogenesis,possible signaling mechanisms and treatment options for GCC.Perspectives for future research are discussed.The tumor likely arises from pluripotent intestinal epithelial crypt base stem cells.Previous findings of Notch signaling as a tumor suppressor in Neuroendocrine tumors may have a similar role in this tumor too.Loss of Notch signaling may be the driver mutation with other successive downstream mutations likely favors them into progressing and behavior similar to poorly differentiated adenocarcinoma with minimal neuroendocrine differentiation.A multidisciplinary approach is suggested for optimal outcomes.Surgery remains the main treatment modality.Simple appendectomy may be sufficient in early stages while right hemicolectomy is recommended for advanced tumors.Cytoreductive surgery with heated intraperitoneal chemotherapy may improve survival in a select few with metastatic peritoneal disease.These tumors have an unpredictable behavior even in early stages and local recurrence and delayed metastases may be seen.Lifelong surveillance is warranted.
