Construction and Practice of Experimental System for Undergraduate Teaching Based on CRISPR/Cas9 Gene Editing Technology / 中国生物化学与分子生物学报

CRISP R/Cas9 is an emerging gene editing technique, which plays an important role in life science research.It is of great significance to introduce this cutting-edge scientific technique into the experimental teaching for undergraduates.Therefore, we established an undergraduate experiment system based on CRISPR/Cas9 technology.This experiment system focuses on the application of CRISPR/Cas9- mediating gene editing in mammalian cells.An engineered mouse embryonic fibroblast which genome were inserted with fluorescein mCherry gene was selected as the experimental model, and called STO-82.Firstly, sgRNAs targeting mCherry gene were designed to construct CRISPR-Cas9/sgRNA co-expression plasmids.After being confirmed by sequencing, they were transfected into STO-82 cells.Two groups of cells with mCherry negative and positive signals were detected by fluorescence-activated cell sorting.Single cells with negative fluorescence were separated and then cultivated to become monoclonal cells.The mutation status of mCherry gene in monoclonal cell lines was detected by sequence analysis.The result showed that there were mutations of insertion or deletion at target sites, indicating that the experimental system was successfully established.Therefore, this comprehensive experiment is comprised of sgRNA design, construction of CRISPR-Cas9/sgRNA co-expression plasmids, cell transfection, cell sorting, monoclonal cell cultivation and sequence analysis.This experiment system is used for experimental teaching for senior undergraduates.Teaching practice can either be decomposed into content modules or be taken as a whole program in light of actual situation.In the teaching practice at 3 classes (13 groups in total, two students every group), which adopted the model of small-class teaching (about 10 students per class), the majority completed the content modules and the expected outcomes were achieved.Through the design and teaching practice of this experiment system, the students acquire a deeper understanding for the principle and experimental procedure of CRISPR/Cas9 technology, an enhanced experimental ability and rigorous scientific thinking and also some knowledge in the risk of its medical application.

Improved synthesis process of optical-magnetic bimodal probe of Gd-4, 7-Bis-carboxymethyl-10-(2-fluoresceinthioureaethyl)-1, 4, 7, 10-tetraaza-cyclododec-1-yl-acetic acid complexes / 北京大学学报(医学版)

OBJECTIVE@#To improve the methods to synthesize and purify of optical-magnetic bimodal molecular probe of Gd-[4, 7-Bis-carboxymethyl-10-(2-fluorescein thioureaethyl)-1, 4, 7, 10-tetraaza-cyclododec-1-yl]-acetic acid complexes.@*METHODS@#Target compound (7), optical-magnetic bimodal molecular molecular probe, was synthesized by the use of 1, 4, 7, 10-tetraazacyclododecane (1) as starting material via substitution reaction, hydrolysis reaction, coupling reaction and complexation reaction with metal.@*RESULTS@#The synthetic route of Gd-[4, 7-Bis-carboxymethyl-10-(2-fluoresceinthioureaethyl)-1, 4, 7, 10-tetraaza-cyclododec-1-yl]-acetic acid complexes was improved. The optical-magnetic bimodal molecular probes were synthesized by substitution reaction, hydrolysis reaction, coupling reaction and complex reaction with metal respectively. For the improved route, the total yield could reach 34.6% which was higher than the original route (18.0%). The structures of those compounds were identified by 1H nuclear magnetic resonance, 13C nuclear magnetic resonance, and mass spectrometry. The improved route could avoid the uncontrollable disadvantage of the substitution reaction, this process could reduce the formation of impurities and made the purification process easier, and in the aspect of purification and separation, the preparative high-performance liquid chromatography with less sample loading and high cost was improved to a column chromatography with many sample loads and being easy to operate. Therefore, the use of column chromatography could be more conducive to mass production of the optical-magnetic bimodal molecular molecular probe.@*CONCLUSION@#The improved synthetic route improves the controllability of the reaction conditions and makes it easier to purify and separate the compounds. At the same time, the improved synthetic route can increase the total yield significantly. The optical-magnetic bimodal molecular probe can combine the living magnetic resonance imaging with the in vitro optical imaging to realize the dual synchronous detection of magneto-optics, so that the detection results of the living magnetic resonance imaging and the in vitro optical imaging are mutually verified. In other words, this synthetic optical-magnetic bimodal molecular probe will make the experimental results more accurate and reliable. In subsequent biological experimental studies, the optical-magnetic bimodal molecular probe can be applied to related research of brain structure and function, and the probe can be used for the brain-related diseases researches, such as brain tumors. after intravenous administration, and thus the optical-magnetic bimodal molecular probe can play an important role in medical treatment of brain tumors and cerebrovascular diseases.

Investigation and application of Dendrobium resources in Qinba Mountains with a discussion on industrial poverty alleviation model / 中国中药杂志

The Dendrobium species are rare and endangered medicinal plants, and it is difficult to investigate their wild resources with conventional methods because of typical epiphytic herbaceous. We explored Dendrobium resources(include culture resource) of Qinba Mountains and the boundary Mountain area in Hubei, Chongqing using the methods of literatures and field investigation, and found that the cultural base of Dendrobium were profound in Qinba Mountains region. Furthermore, its germplasm resources of Dendrobium were established for the first time in Wanzhou Luotian town. In case the advantages of local rock resources and poverty alleviation demand, we have actively carried out the cultivating mode of Dendrobium which grow on rock, and the poverty alleviation model of local characteristic Dendrobium industry were established preliminarily. Our application case can provide reference for the mining and transformation of traditional Chinese medicine resources census results.

Original materials of traditional Chinese medicinal names of "Jinchai" and "Jinchai Shihu" based on vegetative morphology / 中国中药杂志

"Jinchai Shihu" were called Jinchai and recoded in "Taishang Zhouhou Yujingfang" of Tang Dynasty, which first clearly documented the name of Shihu in complex Dendrobium medicines and were condiered as superior medicinal articles. Morphological features are one of the naming principles for Chinese medicines. In this paper, botanical origin plants under the names of "Jinchai" and "Jinchai Shihu" were investigated. Based on documents from the local Chronicles and historical accounts, the Chinese characters of Jinchai have the distinctive features of gold color and two hair clasps. Moreover, the hair clasps are usually cylindrical in shape with uniform thickness in middle and upper part, and tapers off to the foot. And its bottom part style is simple and head part is complex. Thus we speculated the herbal "Jinchai" and "Jinchai Shihu" should have similar morphologic features as Chinese characters of Jinchai, including golden color and hairpin shape of stems without braches, short and solid sterm. After comparing the dried vegetative morphology of 10 common medicinal Dendrobium species, we suggested that of Dendrobium flexicaule matches well with the morphological features from historical herbal records.

Efficacy of intravitreal Conbercept injection for macular edema secondary to macular branch retinal vein occlusion / 国际眼科杂志(Guoji Yanke Zazhi)

AIM:To evaluate the efficacy and safety of intravitreous injection of Conbercept for macular edema secondary to macular branch retinal vein occlusion(MBRVO).METHODS:Clinical records of 19 patients(19 eyes) who were diagnosed MBRVO with macular edema (ME) in our hospital,from July 2015 to September 2016 were retrospectively analysed.All patients were treated with an intravitreal dose of 0.5mg or 0.05mL conbercept ophthalmic injection by the 3+ pro re nata (PRN) project.All patients were evaluated by best corrected visual acuity (BCVA),central retinal thickness(CRT),the number of the injection,and the complications.RESULTS:During the 1,2,3 and 6mo after treatment the mean BCVA all improved significantly(P＜0.01),and the mean CRT reduced statistical significantly(P＜0.01);3 eyes had refractory ME,and micro-aneurysm leakage were identified by FFA,and the ME was suppressed effectively after local laser photocoagulation.No complications,such as secondary vitreous haemorrhage,retinal detachment,persistent high intraocular pressure and endophthalmitis were observed during subsequent follow-up.CONCLUSION:Intravitreous injection of conbercept for macular edema secondary to MBRVO may reduce macular edema and improve visual acuity effectively and safely in the short term.

Application of elastic intramedullary nail in the treatment of primary aneurysmal bone cyst of extremities / 中华骨科杂志

Objective To compare the treatment effect and study the efficacy of the elastic intramedullary nails for the treatment of primary aneurysmal bone cysts in limbs.Methods Twenty one children with primary aneurysmal bone cystsadmitted to and treated in Shanghai Children's Hospital from 2012 January to 2016 June were studied retrospectively.There were 18 males and 3 females,with overall age of 1-14 years old and median age of 5 years old.The lesions were mainly located in the femur,tibia,fibula and humerus.They were divided into 2 groups according to the treatment plan.The patients of the experimentgroup received curettage and bone grafts with high-speed burring and elastic intramedullary nail.The patients of the control group received curettage and bone grafts with high-speed burring.According to the imaging results (Neer grading) and MSTS functional evaluation,the curative effect of the children of the 2 groups were analyzed statistically.Results The cure children were followed up for more than 2 yearswith follow-up time range of 24-48 months (mean value of 32.29±8.22 months).Five patients had a recurrence,with mean postoperative recurrence time of 10±2.66 months.A total of 5 patients of the control group perceived curettage and bone grafts with high-speed burring and a total of 16 patients of the experimentgroup received curettage and bone grafts with high-speed burring andelastic intramedullary nail.According to the imaging results (Neer grading) and MSTS function evaluation,Neer grading was 3 cases for grade Ⅰ,0 case for grade Ⅱ,1 case for grade Ⅲ and 12 cases for grade Ⅳ in the experiment groups.That was 1 case for grade Ⅰ,1 case for grade Ⅱ,2 cases for grade Ⅲ and 1 case for grade Ⅳ in the control group.The ABC recurrence rate of experimental group (18.8％) was lower than matched group (40％),while there was no significant difference.We found that MSTS functional scores of experimental group was significantly higher than the matched group (28.88±2.22 vs 20.80± 10.38,t=3.058,P=0.006).According to postoperative MSTS functional evaluation,we found that even the patients in the study group with recurrencewere also able to participate in physical exercises,without further occurrence of pathological fracture and the postoperative life quality was greatly improved.Conclusion Curettage and bone grafts with high-speed burring andelastic intramedullary nail can significantly reduce the recurrence rate of primary aneurysmal bone cysts in the four limbs.Meanwhile the MSTS function evaluation of the recurrence cases in this group all achieve to a satisfactory level.The reason of recurrence may be correlated to patient age and the distance between the focus and epiphyseal plate.Therefore,an appropriate treatment protocol is helpful in reducing the relapse rate and improving the patients' postoperative living quality.

Serum IL-17 and eotaxin levels in asthmatic patients with allergic rhinitis

Objective: To investigate the serum levels of Interleukin [IL]-17 and eotaxin levels and the relationship between serum IL-17, eotaxin and pulmonary function in asthmatic patients with allergic rhinitis

Methods: Serum IL-17 and eotaxin levels in asthmatic patients with allergic rhinitis during attacking and remission and in healthy control subjects were measured using enzyme-linked immunosorbent assay [ELISA] kits. Then we studied the correlation between the serum IL-17, eotaxin levels and pulmonary function in patients

Results: Serum IL-17 and eotaxin levels were significantly elevated in patients during asthma attack and remission compared with healthy control subjects. These levels in patients during asthma attack were much higher than those during remission. Furthermore, serum IL-17 and eotaxin levels were negatively correlated with pulmonary function in asthmatic patients with allergic rhinitis, respectively

Conclusion: Our findings suggest that IL-17 and eotaxin are important factors in asthma with allergic rhinitis, and the correlation between serum IL-17, eotaxin and lung function possibly lead to improvements in the diagnosis and treatment of asthma with allergic rhinitis and related diseases

Investigation of original materials under traditonal Chinese medicine names of "Jinchai" and "Jinchai Shihu" / 中国中药杂志

To clear from botanical view the original materials under the traditional Chinese medicine names of "Jinchai", "Jinchai Shihu" within the genus Dendrobium of the family Orchidaceae. Combined of different methods including study of historical records from the local chronicles and historical accounts of past event in Hubei, Sichuan, Chongqing, Henan and Shaanxi provinces, interviewing face to face with the old traditional Chinese workers and folk doctors in 20 downtowns and countrysides, such as, Laohekou, Lichuan, Fangxian, Xixia, Neixiang, and Ankang, and collecting a few plants of "Jinchai" for taxonomic identification. The traditional Chinese medicine names of "Jinchai", "Jinchai Shihu" were widely used by the local people from the eastern Chongqing, western Hubei, northeasten Sichuan, southeastern Shaanxi, western Henan. Those two names were frequently found in the local Chronicles and historical accounts of past event, even in the local daily life such as folk songs and stories. The botanical identification results showed that a endemic species of D. flexicaule is the original materials of the traditional Chinese medicine names "Jinchai" and "Jinchai Shihu", and this species are also called "Longtoujin", "Renzijin", "Huanzijin" and "Longtoufengweijin" by the local people. The dried artifactitious specimens of D. flexicaule are traditionally named as "Jinerhuan". The botanic resource plants of the traditional Chinese medicine names of "Jinchai", "Jinchai Shihu" are the endemic species of D. flexicaule that is distributed mainly in central areas of China including eastern Chongqing, western Hubei, northeasten Sichuan, southeastern Shaanxi, and western Henan, rather than D. nobile as referring in both Chinese and English version of Flora of China, and in official recorded serious versions of The Chinese Pharmacopoeia since 1977. In order to avoid confusion in the traditional Chinese medicine dendrobiums industry, the Chinese name of D. nobile is suggested as "Biancao Shihu", which characterized one stem feature of this species, and the traditional Chinese medicine names "Jinchai" or "JinchaiShihu" is suggested to refer to the species D. flexicaule.

Synergistic myoprotection of L-arginine and adenosine in a canine model of global myocardial ischaemic reperfusion injury / 中华医学杂志(英文版)

<p><b>BACKGROUND</b>Endogenous nitric oxide and adenosine increase simultaneously to keep the balance of energy demand and supply when the oxygen supply is insufficient, which suggests that nitric oxide and adenosine might exert a synergistic myoprotection during tissue hypoxia. In this study, we tested this hypothesis utilizing a canine model of prolonged global myocardial ischaemic reperfusion injury.</p><p><b>METHODS</b>In this double blind, controlled study, the hearts of 24 anaesthetized mongrel dogs were arrested for 2 hours with aortic cross clamping and blood cardioplegia. The treatment groups were those supplemented with 2 mmol/L L-arginine (ARG), supplemented with 1 mmol/L adenosine (ADO), ARG + ADO supplemented with both, and no supplementation (control) (n = 6 in each group). Haemodynamics, biochemical indices, adenosine triphosphate (ATP) content and myeloperoxidase activities of myocardium were determined to evaluate myocardial injury. Statistical comparison was performed by two way ANOVA.</p><p><b>RESULTS</b>Although the requirements for inotropic supports were higher, the cardiac outputs were lower in control group than in ARG, ADO and the combination groups. Plasma cardiac troponin I levels were higher and the areas of hydropic changes were larger in control group than in ARG and ADO groups. Combination of arginine and adenosine provided further myoprotection with respect to better cardiac performance, lower release of cardiac troponin I, and smaller areas of hydropic changes compared with ARG and ADO groups. ATP content was higher, but myeloperoxidase activities of myocardium were significantly lower in the combination group than in control, ARG and ADO groups (P < 0.05).</p><p><b>CONCLUSIONS</b>Combination of L-arginine and adenosine provides synergistic myoprotection in a canine model of global myocardial ischaemia. Thus, the combination is recommended when the heart is exposed to a prolonged ischaemia during cardiac surgery.</p>

Effect of High Fat and High Energy Diet on Expression of Sirt1/FOXO1 in White Fat Tissue of Young Rats / 实用儿科临床杂志

OR group,moreover the differences were significant(P0.05),however,both genes in the 2 groups′ expression level significantly were lower than that in control group(Pa

Study on the association of serum matrix metalloproteinase-9 and white blood cell count to type 2 diabetes mellitus with macroangiopathy and the interventing treatment / 中华内分泌代谢杂志

Objective To evaluate the relationship between serum matrix metalloproteinase-9(MMP-9), white blood cell(WBC)count and type 2 diabetes mellitus with macroangiopathy and to investigate the mechanism of the protective effects of rusiglitazone(RSG)on blood vessel.Methods Serum MMP-9 was determined by ELISA in 30 normal controls and 80 type 2 diabetic patients(including 40 cases with macroangiopathy and 40 without maeroangiopathy).WBC count and other clinical parameters were also determined.32 type 2 diabetic patients received RSG(4rag qd)for 12 weeks.All parameters were determined after 4 weeks(17 patients)and after 12 weeks(all patients)to observe the changes in MMP-9,WBC and other parameters.Results In the diabetic patients,serum MMP-9 and WBC were markedly higher as compared with normal controls;and MMP-9 and WBC in patients with macroangiopathy[579(440-949)?g/L,(7.51?1.47)?10~9/L]were higher than those [324(275-423)?g/L,(6.22?0.79)?10~9/L]in the cases without macroangiopathy(P

On expression of VEGF-C and its receptor in breast carcinoma tissue and in peritumoral tissue and their clinic significance / 肿瘤研究与临床

Objective To study the expression of VEGF-C and its receptor in breast carcinoma tissue and in peritumoral tissue,as well as their clinic significance.Methods Immunohistochemistry SP method was used to examine the expression of VEGF-C and VEGFR3 in 70 cases of breast cancer and in its peritu- moral tissue.Results In all 70 cases of breast cancer,the positive expression rate of VEGF-C in breast car- cinoma tissue was 78.6 %,and its rate in peritumoral tissue was 54.3 %.There was a significant stastistic dif- ference between the two groups(P