1.Tissue-SELEX for screening nucleic acid aptamers targeting atherosclerotic tissue
Jianmin CHEN ; Zhimian SHI ; Yanhong LI ; Haiqing ZHAO ; Wenwang LANG ; Zhongcheng MO ; Jiangbo WANG
Chinese Journal of Arteriosclerosis 2025;33(11):937-943
Aim Systematic evolution of ligands by exponential enrichment(SELEX)techniquewas employed to screen and identify nucleic acid aptamers that specifically bind to mouse atherosclerotic pathological tissues,aiming to pro-vide a research foundation for the development of molecular targets and diagnostic reagents for early atherosclerosis.Methods A single-stranded DNA(ssDNA)library with a capacity of 1015~1016 was constructed,which was then subjec-ted to binding-elution(negative selection)with normal mouse vascular tissue slices.The eluted library was subsequently bound to atherosclerotic tissue slices for binding-elution(positive selection).PCR was used to amplify the positive and negative screening products,and agarose gel electrophoresis was used to verify the amplified products.The ssDNA library after multiple rounds of selection was sequenced using T-A cloning and sequencing to obtain the primary structure of the nu-cleic acid aptamers,and the secondary structure was predicted using the Mfold online software.The selected nucleic acid aptamers were labeled with a FAM fluorescent group at the 5'-end and were bound to both positive and negative selection tissue slices,with fluorescence intensity observed under a fluorescence microscope.Image Pro Plus 6.0 was used to cal-culate the relative average fluorescence intensity to evaluate the binding specificity of nucleic acid aptamers.Results After 8 rounds of selection,agarose gel electrophoresis imaging showed PCR amplification products in the positive selection lanes,while no PCR amplification products were observed in the negative selection lanes,indicating the successful acquisi-tion of a nucleic acid aptamer library that specifically binds to atherosclerotic tissues.Five nucleic acid aptamers were i-dentified by T-A cloning and sequencing,and their predicted secondary structures all had stem-loop structures.Immuno-fluorescence staining verified that five nucleic acid aptamers had different degrees of binding with As blood vessels,and the quantitative results of the relative average fluorescence intensity showed that nucleic acid aptamer No.11 had the highest relative average fluorescence intensity value,which can be used as a candidate nucleic acid aptamer for subsequent re-search.Conclusion Specific nucleic acid aptamers that bind to atherosclerotic vesselswere successfully obtained,providing a research foundation for further screening of early molecular targets of Asand developing in vivo early diagnostic reagents.
2.Tissue-SELEX for screening nucleic acid aptamers targeting atherosclerotic tissue
Jianmin CHEN ; Zhimian SHI ; Yanhong LI ; Haiqing ZHAO ; Wenwang LANG ; Zhongcheng MO ; Jiangbo WANG
Chinese Journal of Arteriosclerosis 2025;33(11):937-943
Aim Systematic evolution of ligands by exponential enrichment(SELEX)techniquewas employed to screen and identify nucleic acid aptamers that specifically bind to mouse atherosclerotic pathological tissues,aiming to pro-vide a research foundation for the development of molecular targets and diagnostic reagents for early atherosclerosis.Methods A single-stranded DNA(ssDNA)library with a capacity of 1015~1016 was constructed,which was then subjec-ted to binding-elution(negative selection)with normal mouse vascular tissue slices.The eluted library was subsequently bound to atherosclerotic tissue slices for binding-elution(positive selection).PCR was used to amplify the positive and negative screening products,and agarose gel electrophoresis was used to verify the amplified products.The ssDNA library after multiple rounds of selection was sequenced using T-A cloning and sequencing to obtain the primary structure of the nu-cleic acid aptamers,and the secondary structure was predicted using the Mfold online software.The selected nucleic acid aptamers were labeled with a FAM fluorescent group at the 5'-end and were bound to both positive and negative selection tissue slices,with fluorescence intensity observed under a fluorescence microscope.Image Pro Plus 6.0 was used to cal-culate the relative average fluorescence intensity to evaluate the binding specificity of nucleic acid aptamers.Results After 8 rounds of selection,agarose gel electrophoresis imaging showed PCR amplification products in the positive selection lanes,while no PCR amplification products were observed in the negative selection lanes,indicating the successful acquisi-tion of a nucleic acid aptamer library that specifically binds to atherosclerotic tissues.Five nucleic acid aptamers were i-dentified by T-A cloning and sequencing,and their predicted secondary structures all had stem-loop structures.Immuno-fluorescence staining verified that five nucleic acid aptamers had different degrees of binding with As blood vessels,and the quantitative results of the relative average fluorescence intensity showed that nucleic acid aptamer No.11 had the highest relative average fluorescence intensity value,which can be used as a candidate nucleic acid aptamer for subsequent re-search.Conclusion Specific nucleic acid aptamers that bind to atherosclerotic vesselswere successfully obtained,providing a research foundation for further screening of early molecular targets of Asand developing in vivo early diagnostic reagents.
3.60Co-γ Radiation-induced Population Variation and Cluster Analysis of M1 Generation of Andrographis paniculata
Yumiao SU ; Xiaohan LIU ; Qin DU ; Zhipeng LI ; Zhimian SHI ; Na LI ; Ruihua HUANG
China Pharmacy 2019;30(24):3399-3404
OBJECTIVE: To study the effects of 60Co-γ radiation on the botanical traits and property of Andrographis paniculata, and to screen suitable irradiation dose. METHODS: The seeds of A. paniculata were irradiated by 60Co-γ rays with different irradiation doses(0,10,20,50,100,200,300 Gy). The botanical traits indexes of A. paniculata as seed germination rate, root length, seedling rate, seedling height, leaf area, fresh weight, dry weight, stomata number of lower epidermis of leaf, and its property indexes as the contents of andrographolide, dehydrated andrographolide and chlorophyll, activity of T-SOD enzyme and CuZn-SOD enzyme, were determined after radiation. The coefficient of variation (CV) was calculated. The linear regression analysis was performed for seedling rate, and medial lethal dose was calculated. The correlation analysis was performed between the parameters of botanical trait and quality property with irradiation dose. Cluster analysis was conducted for M1 generation of A. paniculata in different irradiation dose groups by connection method combined with squared euclidean distance. RESULTS: Different irradiation doses showed different effects on botanical traits and property of A. paniculata. According to the average value of CV, the index of botanical traits was ranked as leaf area > fresh weight > dry weight > plant height > root length > stomata number > seedling rate > germination rate; among different irradiation dose groups, the coefficient of variation was ranked as 50 Gy>200 Gy>100 Gy>20 Gy>10 Gy>300 Gy>0 Gy. According to the average value of CV, the index of property was ranked as dehydrated andrographolide content>andrographolide content>chlorophyll content>CuZn-SOD enzyme activity>T-SOD enzyme activity; among different irradiation dose groups, the coefficient of variation was ranked as 100 Gy>50 Gy>200 Gy>20 Gy>10 Gy>300 Gy>0 Gy. The medial lethal dose was 195.10 Gy. According to the botanical traits, M1 generation of A. paniculata of 7 dose groups could be divided into 4 types. According to the property, M1 generation of A. paniculata of 7 dose groups could be divided into 3 types. CONCLUSIONS: The suitable irradiation dose interval for irradiating A. paniculata is 50-200 Gy.
4.Effects of 3 Kinds of Rhizobia on Germination of Andrographis paniculata Seed under Drought and Salt Stress Condition
Zhimian SHI ; Qin DU ; Yumiao SU ; Xiaohan LIU
China Pharmacy 2019;30(22):3104-3108
OBJECTIVE: To study the effects of Sinorhizobium fredii, Rhizobium radiobacter and Azorhizobium caulinodans on germination of Andrographis paniculata seed under drought and salt stress condition. METHODS: Drought and salt stress models of A. paniculata seed were established with PEG-6000 (called “PEG” for short)and NaCl respectively. The effects of different concentrations of PEG (0.05, 0.10, 0.15, 0.20, 0.25 g/mL) and NaCl (50, 100, 150, 200 mmol/L) on the germination indicators (germination rate, germination potential, germination index, vigor index) of A. paniculata seed were investigated by the method of germination on dish paper. The seeds were soaked with S. fredii, R. radiobacter and A. caulinodans, and then germination indicators were investigated under the condition of drought and salt stress. RESULTS: Drought and salt stress models of A. paniculata seed were established with 0.15 g/mL PEG solution and 50 mmol/L NaCl solution, respectively. S. fredii pretreatment could significantly improve all the germination indicators of A. paniculata seed under drought stress condition, as well as germination index and vigor index of it under salt stress condition, but significantly reduce germination rate and germination index of it under salt stress condition (P<0.05). R. radiobacter treatment could significantly improve all the germination indicators of A. paniculata seed under drought stress condition, as well as germination potential, germination index and vigor index of it under salt stress condition (P<0.05). A. caulinodans pretreatment could significantly improve germination rate, germination index and vigor index of A. paniculata seed under drought stress condition, as well as germination rate and germination potential of it under salt stress condition (P<0.05). CONCLUSIONS: Three kinds of Rhizobia can improve germination ability of A. paniculata seed under drought or salt stress condition to different degrees. This research can provide technical support for planting and cultivation of A. paniculata under drought condition, and using saline-alkali soil as A. paniculata plantation area.
5.Identification of Ligustrum sinense Lour. And Jasminum elongatum (Bergium) Wild.
Zhimian SHI ; Fang TIAN ; Qin DU ; Zhenhua WANG
Journal of Guangzhou University of Traditional Chinese Medicine 2017;34(3):424-431
Objective To identify the Ligustrum sinense Lour.and Jasminum elongatum(Bergium) Wild.from the macroscopic appearance and microscopic features.Methods Fresh samples of stems,leaves,flowers and fruits of the two kinds of medicinal plants were harvested.Stereoscopy was used for the observation of macroscopic appearance of Ligustrum sinense Lour.and Jasminum elongatum (Bergium) Wild.,and the microscopy was used for the examination of their microscopic features of stem and leaf cross section,tear film and the powder of leaf upper and lower epidermis.Results Original plant characteristics of the two kinds of species were as follows:Ligustrum sinense Lour.had a racemes and funnel-shaped corolla,while Jasminum elongatum (Bergium)Wild.had a cymes and salverform corolla.Microscopic identification results were as follows:the stem pith of Ligustrum sinense Lour.was smaller,and the stem pith ofJasminum elongatum(Bergium) Wild.was bigger;Ligustrum sinense Lour.stem powder had less pit or texture in the sclereids,and had reticulate vessels,and Jasminum elongatum(Bergium) Wild.stem powder had apparent pit or texture in the sclereids,and had spiral vessels.Conclusion The results will provide a basis for the identification,exploitation and utilization of Ligustrum sinense Lour.and Jasminum elongatum(Bergium) Wild.

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