Objective:To explore the methods of fertility preservation in prepubertal patients with thalassemia major (TM), and to provide further data support for the fertility preservation in prepubertal patients with hematologic diseases.Methods:A case of a prepubertal patient with TM who required urgent hematopoietic stem cell transplantation (HSCT) receiving ovarian tissue cryopreservation (OTC) and in vitro maturation (IVM) to preserve fertility was reported, and the timing, the indications and strategies of fertility preservation in prepubertal girls with thalassemia were discussed in combination with related literature. Results:After ovarian tissue extraction, a total of 24 cumulus-oocyte complexes (COCs) and 11 ovarian cortex pieces were obtained through puncture and aspiration. After IVM for 48 h, a total of 9 M Ⅱ oocytes were frozen by vitrification. Conclusion:For the prepubertal girls facing HSCT urgently, cryopreserving ovarian tissue in combination with retrieving immature oocytes followed by IVM can preserve the fertility of patients to the greatest extent in a short period of time, as well as improve the therapeutic effect of fertility preservation in patients.

Objective:To explore the feasibility of autologous transplantation of frozen-thawed ovarian tissue to induce pubertal development in adolescent females.Methods:Before hematopoietic stem cell transplantation in patient with severe β-thalassemia, 11 pieces of ovarian tissue were frozen in the Center of Reproductive Medicine, the Sixth Affiliated Hospital of Sun Yat-sen University in 2019. The patient was diagnosed as premature ovarian failure after hematopoietic stem cell transplantation. There were no signs of puberty development and menarche. Orthotopic ovarian tissue transplantation was performed for the patient through laparoscopy, and a total of 5 pieces of ovarian tissue were transplanted on January 20, 2022. Postoperatively, we followed up the sex hormone levels, growth and development of the patients and menarche.Results:The patient developed menarche 5 months after ovarian transplantation. The levels of sex hormones showed that follicle-stimulating hormone and luteinizing hormone were significantly decreased, and estradiol levels were significantly increased, indicating that ovarian tissue transplantation was successful, and follicles had begun to recruit and develop. The patient's ultrasonography revealed a markedly enlarged uterus and a thickened endometrium. Antral follicles were detected in the left implantation site of pelvic cavity.Conclusion:Cryopreservation of ovarian tissue is recommended for fertility preservation in prepubertal children. Autologous frozen-thawed ovarian tissue transplantation can induce natural puberty development and restore the reproductive endocrine function in children with ovarian failure, delayed puberty development or even stagnation.

Objective:To explore the methods of fertility preservation in prepubertal patients with thalassemia major (TM), and to provide further data support for the fertility preservation in prepubertal patients with hematologic diseases.Methods:A case of a prepubertal patient with TM who required urgent hematopoietic stem cell transplantation (HSCT) receiving ovarian tissue cryopreservation (OTC) and in vitro maturation (IVM) to preserve fertility was reported, and the timing, the indications and strategies of fertility preservation in prepubertal girls with thalassemia were discussed in combination with related literature. Results:After ovarian tissue extraction, a total of 24 cumulus-oocyte complexes (COCs) and 11 ovarian cortex pieces were obtained through puncture and aspiration. After IVM for 48 h, a total of 9 M Ⅱ oocytes were frozen by vitrification. Conclusion:For the prepubertal girls facing HSCT urgently, cryopreserving ovarian tissue in combination with retrieving immature oocytes followed by IVM can preserve the fertility of patients to the greatest extent in a short period of time, as well as improve the therapeutic effect of fertility preservation in patients.

Objective:To explore the feasibility of autologous transplantation of frozen-thawed ovarian tissue to induce pubertal development in adolescent females.Methods:Before hematopoietic stem cell transplantation in patient with severe β-thalassemia, 11 pieces of ovarian tissue were frozen in the Center of Reproductive Medicine, the Sixth Affiliated Hospital of Sun Yat-sen University in 2019. The patient was diagnosed as premature ovarian failure after hematopoietic stem cell transplantation. There were no signs of puberty development and menarche. Orthotopic ovarian tissue transplantation was performed for the patient through laparoscopy, and a total of 5 pieces of ovarian tissue were transplanted on January 20, 2022. Postoperatively, we followed up the sex hormone levels, growth and development of the patients and menarche.Results:The patient developed menarche 5 months after ovarian transplantation. The levels of sex hormones showed that follicle-stimulating hormone and luteinizing hormone were significantly decreased, and estradiol levels were significantly increased, indicating that ovarian tissue transplantation was successful, and follicles had begun to recruit and develop. The patient's ultrasonography revealed a markedly enlarged uterus and a thickened endometrium. Antral follicles were detected in the left implantation site of pelvic cavity.Conclusion:Cryopreservation of ovarian tissue is recommended for fertility preservation in prepubertal children. Autologous frozen-thawed ovarian tissue transplantation can induce natural puberty development and restore the reproductive endocrine function in children with ovarian failure, delayed puberty development or even stagnation.

Objective: To analyze the risk factors of varicella zoster virus(VZV) infection after allogeneic hematopoietic stem cell transplantation(allo-HSCT) in children, and to provide reference for the diagnosis, monitoring and prophylaxis of VZV infection after allo-HSCT. Methods: A total of 367 patients, who underwent allo-HSCT in Pediatric Transplantation Center of Nanfang Hospital Affiliated to Southern Medical University from January 2012 to June 2015 were collected.Clinical characteristics and risk factors of the patients complicated with VZV after allo-HSCT were retrospectively analyzed. Results: Thirty-four patients (9.26%) were complicated with VZV infection after allo-HSCT.The median onset time was 96.5 d(19-326 d). Two of 34 patients relapsed 3 times, 1 case of them relapsed twice, 3 cases of them relapsed once, and 4 cases of 34 patients were complicated with VZV encephalitis.All cases were treated with antivirus drugs, infusion of immunoglobulin, reduction of immunosuppressant dosages, with external use of Acyclovir ointment.The median therapy time was 13 days (7-28 days). All of their herpes subsided, and neurological symptoms such as headache, vomiting and convulsion disappeared.VZV-DNA both in blood and cerebrospinal fluid turned negative.No patient had herpetic dissemination and visceral involvement, and no one died directly of VZV infection.Results indicated that age(χ²=6.863, P=0.009), underlying disease(χ²=14.793, P=0.022), type of HSCT(χ²=14.459, P=0.001) and resource of stem cell (χ²=20.585, P=0.002) were significant risk factors for VZV infection after allo-HSCT, while sex (χ²=0.106, P=0.745) and antithymocyte globulin in conditioning regimen(χ²=0.010, P=0.921) had no relation to it. Conclusions VZV infection mainly occur within 12 months after allo-HSCT and is prone to be complicated with VZV encephalitis.The effect of Acyclovir is good.Monitoring and prophylaxis of VZV infection after allo-HSCT should be strengthened in children with high risk factors.

Objective To observe the expression changes ofmiR-34a and its related target genes sirtuin 1 (Sirtl) and nuclear factor-E2-related factor 2 (Nrf2) in subependymal zone (SVZ) of rats after cerebral ischemia,and to explore the effect of miR-34a on proliferation of endogenous neural stem cells (NSCs) after cerebral ischemia.Methods Forty SD rats were randomly divided into control group,cerebral ischemia one d group,cerebral ischemia three d group and cerebral ischemia 7 d group (n=10).Middle cerebral artery occlusion models were established by thread embolization in the latter three groups,and the SVZs were taken one,three and 7 d after model making,respectively.Immunofluorescence staining was used to detect the expression of nestin,real-time PCR was used to detect the mRNA expressions ofmiR-34a,Sirtl and Nrf2,and Western blotting was used to detect the protein expressions of Sirt1 and Nrf2.Results (1) In SVZs of cerebral ischemia one d group,cerebral ischemia three d group and cerebral ischemia 7 d group,the number of nestin positive cells increased gradually ([2.013±0.526],[4.821 ±1.154],and [6.394±1.027]) cells/filed,with statistical differences (P＜0.05).(2) In SVZs of cerebral ischemia one d group,cerebral ischemia three d group and cerebral ischemia 7 d group,the expression levels of miR-34a mRNA increased gradually (0.295±0.145,0.701 ±0.075,and 1.136±0.018),the Sirt1 mRNA expression levels showed a downward trend (0.835±0.024,0.620±0.133,and 0.278±0.110),the Nrf2 mRNA expression levels obviously decreased (1.032±0.256,0.833±0.187,and 0.630±0.123),the Sirt1 protein expression levels showed a downward trend (0.832±0.018,0.731±0.156,and 0.454±0.132),and the Nrf2 protein expression levels obviously decreased (1.003±0.133,0.813±0.111,and 0.671 ±0.071),with statistically significant differences (P＜0.05).(3)Statistical correlation analysis showed that the number of nestin positive cells was positively correlated with miR-34a mRNA expression level and negatively correlated with Sirt1 and Nrf2 mRNA and protein expression levels after cerebral ischemia (r=0.887,P=0.003;r=-0.746,P=0.005;r=-0.521,P=0.013;r=-0.344,P=0.025;r=-0.863,P=0.000).Conclusions MiR-34a may regulate the proliferation of NSCs through Sirt1/Nrf2 pathway after cerebral ischemia.

Objective CRISPR/Cas9 genome-editing technique provides an novel method for whole genome editing in eukaryotic cells.Recently,we found that gene subtype library with smaller size and focused pur-pose is more economical and practical. In this study,we aimed to target kinases,a group of pivotal cell signal transducers,to construct a kinase knock-out library using CRISPR/Cas9 technique.The construction strategy wll al-so be discussed. Methods 10 sgRNA was designed for each kinase target.After oligo pool synthesis by semicon-ductor chip,the oligos were eluted from the chip. The oligo templates were amplified and cloned into Cas9 vector and transformed into Stble3 competent cells.Monoclonal colonies were selected for DNA sequencing. Results(1) GO analysis of 507 cell kinases showed that the cell kinases took part in a wide range of cell signaling.(2)The sgRNA pool with about 140 bp in length was successfully amplified by using oligo pool as the template and univer-sal PCR primers.(3)In 40 identified library clones,34 clones were sequenced successfully. Among them,the DNA sequencing results of 25 samples were completely consistent with the designed target sequences.But there are some mutations in the primers of 9 samples.Failure in bacteria shaking,DNA sequencing and other factors were ex-isted in the other clones. Conclusion The CRISPR/Cas9 kinase knock-out library can be widely used for screen-ing the important kinases which may mediate cell proliferation,metastasis,drug resistance and autophagy.This li-brary will play an important role in clarifying the development of disease associated with kinases.

Objective To explore the relationship between prevalence of Kashin-Beck disease (KBD) and ecological environment, and to broaden the perspective of KBD etiology. Methods In 37 counties of KBD areas in Gansu Province, information about the ecological environment and implementation situation of control measures (altitude, temperature, rainfall, evapo ration, frost free period, annual sunshine hours, population density, per capita income, the proportion of staple food, returning farmland to forest, to forestry and replant crops) and X-ray detection rate of KBD of 7-12-year-old children in 2012 - 2014 was collected. Using four quantile regression method, the regression model was introduced to analyze the 11 ecological factors which related to the pathogenesis of KBD. The effect of three points on X-ray detection rate of KBD was estimated. Results The X-ray detection rate of KBD was independent of altitude, temperature, evaporation, population density, per capita income and cash crops, and was dependent of rainfall, frost free period, annual sunshine hours, the staple food purchase ratio, and returning farmland to forest and grassland. No matter where in any place numbered, the higher rainfall (measure value:0.003 3 to 0.006 4), the longer frost free period (measure value: 0.029 2 to 0.043 8), the longer annual sunshine hours (measure value:0.001 6 to 0.001 8), and the higher staple food purchase ratio (measure value:0.019 7 to 0.027 6), the higher risk of X-ray detection rate of KBD; the higher returning farmland to forest and to grassland, the lower risk of X-ray detection rate of KBD (measure value: - 0.037 2 to - 0.013 3). Conclusion The X-ray detection rate of KBD is closely related to local ecological environment.

Objective To grasp the epidemic features of Kaschin-Beck disease in different ecotypic areas in Gansu Province,in order to provide ecological basis for Kashin-Beck disease control.Methods Totally 37 counties with Kashin-Beck disease were divided into four ecological areas,villages with historical serious condition in township and townships with historical serious condition in county were investigated.Clinical examination and Xray of right hand of all 7-12-year-old children in the villages were carried out,while investigation of prevention and control measures was performed.Results Among 27 966 children from four ecological areas,the clinical detection rate was 0.05％ (14/27 966) and the X-ray positive rate was 1.26％ (353/27 966),metaphyseal rate was 1.25％ (350/27 966),bone-side positive rate was 0.01％ (3/27 966) and no case of epiphyseal and carpal were discovered.The clinical detection rate in children in the four ecological areas was not statistically significant (x2() =7.757,P ＞0.05),the Loess Plateau-gully region of Longdong [0.09％ (10/11 604)] ＞ the Anyon area of Qinling of Longnan [0.04％ (3/7 969)] ＞ Alpine grassland meadow area of Gannan [0.02％ (1/4 021)] ＞ the Loess Plateau-hills region of Longzhong [0 (0/4 372)].The detected rate of X-ray in four ecological areas was statistically significant (x2 =18.133,P ＜ 0.05),the Anyon area of Qinling of Longnan [1.49％ (119/7 969)] ＞ the Loess Plateau-gully region of Longdong [1.41％ (164/11 604)] ＞ the Loess Plateau-hills region of Longzhong [0.89％ (39/4 372)] ＞ Alpine grassland meadow area of Gannan [0.77％ (31/4 021)].Comprehensive prevention and control measures on Kaschin-Beck disease were different in different ecotypic areas.Relocation,long-distance education and selenium supplement measures had not been implemented in the four ecological areas.Conclusion The epidemic situation of Kaschin-Beck disease is different in different ecotypic areas in Gansu Province,which maybe related to ecological environment,but is under a state of control.

Objective To evaluate the correlation between osteophytes size and lower limb alignment in the knees of patients with Kaschin-Beck disease (KBD).Methods A total of 300 clinically diagnosed patients with KBD were X-rayed on knee-joints which ranged from the distal half of femur to proximal half of tibia.Meanwhile some related parameters in the X film with the anteroposterior position (including osteophytes length,femorotibial angle,femorotibial joint space ratio of inner side to outer side) were measured by DICOM 2.0,a software of medical graphic measuring,then followed by calculating the osteophyte spur index.The association between femorotibial angle,femorotibial joint space ratio and osteophytes spur index was evaluated by Pearson correlation test.Results The average of femorotibial angle of all the tested knee-joints was (165.97 ± 4.02)°,which positively correlated with both the osteophyte spur index of the medial femoral condyle [(6.54 ± 3.12)％,correlation coefficient (r) =0.524,P＜0.01] and the osteophyte spur index of medial tibil plateau [(7.14 ± 3.40)％,r =0.578,P ＜0.01].The femorotibial joint space ratio was 0.61 ± 0.13,which positively correlated with both the osteophyte spur index of medial femoral condyle (r =0.531,P ＜0.01) and that of the medial tibil plateau (r =0.563,P ＜0.01).Conclusions The results of this study indicate that there is a positive correlation between lower limb alignment and osteophyte size of both the medial femoral condyle and the medial tibial plateau.This finding may be evidenced by the fact that the changes of lower limb biomechanics may contribute to formation and development of osteophytes in the kneejoint.