[Changes in Plasma Amyloid-ß Level and Their Relationship With White Matter Microstructure in Patients With Mild Cognitive Impairment].

Objective To investigate the changes in plasma amyloid-ß (Aß) level and their relationship with white matter microstructure in the patients with amnesic mild cognitive impairment(aMCI) and vascular mild cognitive impairment (vMCI).Methods A total of 36 aMCI patients,20 vMCI patients,and 34 sex and age matched healthy controls (HC) in the outpatient and inpatient departments of the First Affiliated Hospital of Anhui Medical University were enrolled in this study.Neuropsychological scales,including the Mini-Mental State Examination,the Montreal Cognitive Assessment,and the Activity of Daily Living Scale,were employed to assess the participants.Plasma samples of all the participants were collected for the measurement of Aß42 and Aß40 levels.All the participants underwent magnetic resonance scanning to obtain diffusion tensor imaging (DTI) data.The DTI indexes of 48 white matter regions of each individual were measured (based on the ICBM-DTI-81 white-matter labels atlas developed by Johns Hopkins University),including fractional anisotropy (FA) and mean diffusivity (MD).The cognitive function,plasma Aß42,Aß40,and Aß42/40 levels,and DTI index were compared among the three groups.The correlations between the plasma Aß42/40 levels and DTI index of aMCI and vMCI patients were analyzed.Results The Mini-Mental State Examination and the Montreal Cognitive Assessment scores of aMCI and vMCI groups were lower than those of the HC group (all P<0.001).There was no significant difference in the Activity of Daily Living Scale score among the three groups (P=0.654).The plasma Aß42 level showed no significant difference among the three groups (P=0.227).The plasma Aß40 level in the vMCI group was higher than that in the HC group (P=0.014),while it showed no significant difference between aMCI and HC groups (P=1.000).The plasma Aß42/40 levels in aMCI and vMCI groups showed no significant differences from that in the HC group (P=1.000,P=0.105),while the plasma Aß42/40 level was lower in the vMCI group than in the aMCI group (P=0.016).The FA value of the left anterior limb of internal capsule in the vMCI group was lower than those in HC and aMCI groups (all P=0.001).The MD values of the left superior corona radiata,left external capsule,left cingulum (cingulate gyrus),and left superior fronto-occipital fasciculus in the vMCI group were higher than those in HC (P=0.024,P=0.001,P=0.003,P<0.001) and aMCI (P=0.015,P=0.004,P=0.019,P=0.001) groups,while the MD values of the right posterior limb of internal capsule (P=0.005,P=0.001) and left cingulum (hippocampus) (P=0.017,P=0.031) in the aMCI and vMCI groups were higher than those in the HC group.In the aMCI group,plasma Aß42/40 level was positively correlated with FA of left posterior limb of internal capsule (r=0.403,P=0.015) and negatively correlated with MD of the right fonix (r=-0.395,P=0.017).In the vMCI group,plasma Aß42/40 level was positively correlated with FA of the right superior cerebellar peduncle and the right anterior limb of internal capsule (r=0.575,P=0.008;r=0.639,P=0.002),while it was negatively correlated with MD of the right superior cerebellar peduncle and the right anterior limb of internal capsule (r=-0.558,P=0.011;r=-0.626,P=0.003).Conclusions Plasma Aß levels vary differently in the patients with aMCI and vMCI.The white matter regions of impaired microstructural integrity differ in the patients with different dementia types in the early stage.The plasma Aß levels in the patients with aMCI and vMCI are associated with the structural integrity of white matter,and there is regional specificity between them.

Risk factors for relapse and nomogram for relapse probability prediction in patients with minor ischemic stroke.

BACKGROUND: The identification of risk factors for recurrence in patients with minor ischemic stroke (MIS) is a critical medical need. AIM: To develop a nomogram for individualized prediction of in-hospital recurrence in MIS patients. METHODS: Based on retrospective collection, a single-center study was conducted at the First Affiliated Hospital of Anhui Medical University from January 2014 to December 2019. Univariate and multivariate logistic regression analyses were used to determine the risk factors associated with MIS recurrence. The least absolute shrinkage and selection operator regression was performed for preliminary identification of potential risk factors. Uric acid, systolic blood pressure, serum total bilirubin (STBL), and ferritin were integrated for nomogram construction. The predictive accuracy and calibration of the nomogram model were assessed by the area under the receiver operating characteristic curve (AUC-ROC) and Hosmer-Lemeshow test, respectively. RESULTS: A total of 2216 MIS patients were screened. Among them, 155 were excluded for intravascular therapy, 146 for unknown National Institutes of Health Stroke Scale score, 195 for intracranial hemorrhage, and 247 for progressive stroke. Finally, 1244 patients were subjected to further analysis and divided into a training set (n = 796) and a validation set (n = 448). Multivariate logistic regression analysis revealed that uric acid [odds ratio (OR): 0.997, 95% confidence interval (CI): 0.993-0.999], ferritin (OR: 1.004, 95%CI: 1.002-1.006), and STBL (OR: 0.973, 95%CI: 0.956-0.990) were independently associated with in-hospital recurrence in MIS patients. Our model showed good discrimination; the AUC-ROC value was 0.725 (95%CI: 0.646-0.804) in the training set and 0.717 (95%CI: 0.580-0.785) in the validation set. Moreover, the calibration between nomogram prediction and the actual observation showed good consistency. Hosmer-Lemeshow test results confirmed that the nomogram was well-calibrated (P = 0.850). CONCLUSION: Our present findings suggest that the nomogram may provide individualized prediction of recurrence in MIS patients.

A Novel Probable Pathogenic PSEN2 Mutation p.Phe369Ser Associated With Early-Onset Alzheimer's Disease in a Chinese Han Family: A Case Report.

The pathogenesis of Alzheimer's disease is complex, and early-onset Alzheimer's disease (EOAD) is mostly influenced by genetic factors. Presenilin-1, presenilin-2 (PSEN2), and amyloid precursor protein are currently known as the three main causative genes for autosomal dominant EOAD, with the PSEN2 mutation being the rarest. In this study, we reported a 56-year-old Chinese Han proband who presented with prominent progressive amnesia, aphasia, executive function impairment, and depression 5 years ago. The 3-year follow-up showed that the patient experienced progressive brain atrophy displayed on magnetic resonance imaging (MRI) and dramatic cognitive decline assessed by neuropsychological evaluation. This patient was clinically diagnosed as EOAD based on established criteria. A heterozygous variant (NM_000447.2: c.1106T>C) of PSEN2 was identified for the first time in this patient and her two daughters. This mutation causing a novel missense mutation (p.Phe369Ser) in transmembrane domain 7 encoded by exon 11 had not been reported previously in 1000Genomes, ExAC, or ClinVar databases. This mutation was predicted by four in silico prediction programs, which all strongly suggested that it was damaging. Our results suggest that this novel PSEN2 Phe369Ser mutation may alter PSEN2 protein function and associate with EOAD.

Hemorrhagic transformation of ischemic cerebral proliferative angiopathy: A case report.

BACKGROUND: Cerebral proliferative angiopathy (CPA) is a rare vascular disease characterized by the presence of diffuse vascular proliferation, progressive vascular hyperflow and vasodilation of multiple vessels in the normal brain parenchyma. Unlike cerebral arteriovenous malformations, CPA has a mixed appearance between that of lesions with cell proliferation and endothelial proliferation. To date, the pathogenesis of CPA is unclear, in which changes induced by cortical ischemia in the elastic layer of the blood supply artery and smooth muscle cells may be involved. CASE SUMMARY: In this article, we retrospectively analyzed a case of hemorrhagic transformation of ischemic CPA diagnosed by digital subtraction angiography and reviewed the related literature for further exploration of its pathogenesis, diagnosis and treatment. CONCLUSION: The information in the present case report may facilitate further clinical research on this cerebrovascular disease.

Contribution of Inflammation and Hypoperfusion to White Matter Hyperintensities-Related Cognitive Impairment.

White matter hyperintensities (WMHs) of presumed vascular origin are one of the most important neuroimaging markers of cerebral small vessel disease (CSVD), which are closely associated with cognitive impairment. The aim of this study was to elucidate the pathogenesis of WMHs from the perspective of inflammation and hypoperfusion mechanisms. A total of 65 patients with WMHs and 65 healthy controls were enrolled in this study. Inflammatory markers measurements [hypersensitive C-reactive protein (hsCRP) and lipoprotein-associated phospholipase A2 (Lp-PLA2)], cognitive evaluation, and pseudocontinuous arterial spin labeling (PCASL) MRI scanning were performed in all the subjects. The multivariate logistic regression analysis showed that Lp-PLA2 was an independent risk factor for WMHs. Cerebral blood flow (CBF) in the whole brain, gray matter (GM), white matter (WM), left orbital medial frontal gyrus [MFG.L (orbital part)], left middle temporal gyrus (MTG.L), and right thalamus (Tha.R) in the patients was lower than those in the controls and CBF in the left triangular inferior frontal gyrus [IFG.L (triangular part)] was higher in the patients than in the controls. There was a significant correlation between Lp-PLA2 levels and CBF in the whole brain (R = -0.417, p < 0.001) and GM (R = -0.278, p = 0.025), but not in the WM in the patients. Moreover, CBF in the MFG.L (orbital part) and the Tha.R was, respectively, negatively associated with the trail making test (TMT) and the Stroop color word test (SCWT), suggesting the higher CBF, the better executive function. The CBF in the IFG.L (triangular part) was negatively correlated with attention scores in the Cambridge Cognitive Examination-Chinese Version (CAMCOG-C) subitems (R = -0.288, p = 0.020). Our results revealed the vascular inflammation roles in WMHs, which may through the regulation of CBF in the whole brain and GM. Additionally, CBF changes in different brain regions may imply a potential role in the modulation of cognitive function in different domains.

Regulatory effects of miR-146a/b on the function of endothelial progenitor cells in acute ischemic stroke in mice.

The study aims to explore how microRNA-146a/b (miR-146a/b) regulates the function of endothelial progenitor cells (EPCs) in acute ischemic stroke in mice. Eighty male SPF C57BL/6J mice were evenly divided into the model-6 h, model-12 h, model-24 h (mice suffered from middle cerebral artery occlusion [MCAO] for 6 h, 12 h and model-24 h) and normal groups. EPCs were transfected and assigned into the control, MCAO, MCAO-miR-146a, MCAO-miR-146b and MCAO-miR-146a/b groups. The qRT-PCR was used to detect miR-146a/b expression in EPCs. Expressions of tumor necrosis factor receptor-associated factor 6 (TRAF6) and interleukin-1 receptor-associated kinase 1 (IRAK1) were detected using western blotting. Cell proliferation and migration of EPCs were testified using CCK-8 assay and scratch test, respectively. Angiogenesis ability of EPCs was observed under microscope. MiR-146a and miR-146b expressions were lower in the model groups than the normal group. There were up-regulated TRAF6 and IRAK1 expressions in the model-6 h, model-12 h and model-24 h groups compared with the normal group. And there were down-regulated TRAF6 and IRAK1 expressions in the MCAO-miR-146a, MCAO-miR-146b and MCAO-miR-146a/b groups than in the MCAO group. Compared with the control group, the proliferation, migration and angiogenesis ability of EPCs were significantly lower in the MCAO group, but higher in the MCAO-miR-146a, MCAO-miR-146b and MCAO-miR-146a/b groups. Besides, the miR-146a/b group showed more enhancement than the MCAO-miR-146a and MCAO-miR-146b groups. MiR-146a/b could down-regulate the TRAF6 and IRAK1 expressions and promote proliferation, migration and angiogenesis ability of EPCs, which was important for recovery of patients with hyperacute ischemic stroke.

Diagnostic Values of Serum Levels of Homocysteine and Uric Acid for Predicting Vascular Mild Cognitive Impairment in Patients with Cerebral Small Vessel Disease.

BACKGROUND This study aimed to investigate the diagnostic values of serum levels of Hcy and UA for predicting vascular mild cognitive impairment (VMCI) in patients with cerebral small vessel disease (SVD). MATERIAL AND METHODS We selected 172 cerebral SVD patients and divided them into a VMCI group and a non-VMCI group. Eighty-six healthy individuals without nervous system diseases were selected as the control group. Enzymatic cycling method was performed to detect serum Hcy and UA levels. Serum levels of folic acid (FOA) and vitamin B12 (VitB12) were detected by chemiluminescence immunoassay. Montreal cognitive assessment (MoCA) was applied to evaluate the cognitive function. The ROC curve was used to evaluate the diagnostic values of serum Hcy and UA levels for predicting VMCI. Logistic regression analysis was used to determine the possible risk factors. RESULTS Compared with the non-VMCI and control groups, serum FOA and VitB12 levels were lower and serum Hcy and UA levels were higher in the VMCI group. AUC values of serum Hcy and UA levels were 0.703 and 0.829, respectively. Serum Hcy and UA levels were negatively correlated with serum FOA and VitB12 levels, total MoCA score, and subscores on visuospatial ability and executive function, on language ability and on delayed recall, and they were positively correlated with serum cholesterol (CH) level. Serum Hcy and UA levels were indicated as risk factors for VMCI in cerebral SVD patients. CONCLUSIONS These results suggest that serum Hcy and UA levels may serve as predictive factors for VMCI in cerebral SVD patients.

Complete mitochondrial genome of Coregonus peled.

The complete mitochondrial genome of Coregonus peled is determined in this study. The mitogenome is 16,736 bp in length and contains 1D-loop region, 2 ribosomal RNA genes, 22 transfer RNA genes and 13 protein-coding genes. The overall base composition of the heavy strand is 26.76% for A, 29.45% for C, 18.10% for G and 25.67% for T. The percentage of G + C content is 47.55 %. This is the first time of the mitochondrial genome sequencing for Coregonus peled.

[Changes of serum bilirubin and uric acid in patients with myasthenia gravis].

OBJECTIVE: To explore the correlations of serum total bilirubin (TBIL), direct bilirubin (DBIL), indirect bilirubin (IBIL) and uric acid (UA) with myasthenia gravis (MG). METHODS: A total of 131 MG patients were selected as MG group and 176 healthy cases as control group. They were enrolled from the Department of Neurology, First Affiliated Hospital, Anhui Medical University between January 2010 and December 2012. And the controls were recruited from a health check-up center. The total serum BIL and UA concentrations were measured by an enzymatic method. All MG patients underwent thymus computed tomography (CT) scanning. RESULTS: The serum levels of TBIL, IBIL and UA in patients with MG ((11.0 ± 0.4, 7.4 ± 0.3, 270 ± 70) µmol/L) were significantly lower than those in healthy control group ((13.0 ± 0.4, 9.6 ± 0.3, 301 ± 60) µmol/L) (P < 0.01). Moreover, these results were consistent when the male and female cohorts were investigated separately. In MG group, females had significantly lower serum TBIL, IBIL and UA levels ( (10.1 ± 3.7, 6.7 ± 2.7, 242 ± 68) µmol/L) than males ((12.2 ± 5.5, 8.4 ± 4.2, 288 ± 73) µmol/L) (P < 0.05-0.01). However, no difference existed when comparing different grades of MG patients according to the modified Osserman classification. Also no significant difference existed between MG patients with thymic abnormalities and those without. In comparison with the subjects in the reference group (total bilirubin ≥ 11.4 µmol/L, UA ≥ 279 µmol/L), the odds ratio (95% CI) for MG patients in the lower tertile (total bilirubin < 11.4 µmol/L, UA < 279 µmol/L) were 1.98 (1.20-3.29) and 2.22 (1.29-3.82) respectively after multivariable adjustment. Serum level of TBIL and UA had positive correlations with creatinine as indicated by Pearson correlation analysis (r = 0.151, P = 0.008; r = 0.301, P = 0.000). Meanwhile, serum level of UA had a negative correlation with high density lipoprotein-cholesterol (r = -0.347, P = 0.000) . CONCLUSION: Decreased serum levels of BIL and UA are closely correlated with MG. As a replacement therapy, administration of BIL and UA or theirs precursors may offer benefits to the MG patients.

[Influence of leukoaraiosis on the cognition of patients with Parkinson disease].

OBJECTIVE: To explore the role of leukoaraiosis (LA) on the cognitive function in patients with Parkinson disease (PD). METHODS: The cohort for this study included 63 patients with PD, whom were divided into 3 groups according to cognitive status:with intact cognition (PD-IC, n = 23), with mild cognitive impairment (PD-MCI, n = 23) and with dementia (PDD, n = 17). All the patients were recruited from the Department of Neurology, First Affiliated Hospital, Anhui Medical University between September 2011 and July 2012. The cognitive functions were evaluated by mini-metal state examination (MMSE), the Cambridge cognitive examination-Chinese version (CAMCOG-C), clinical dementia rating (CDR), clock drawing task (CDT) and verbal fluency test, etc. Depression symptoms were assessed by the geriatric depression scale (GDS) while motor symptoms by the Unified Parkinson's Disease Rating Scale-motor (UPDRS-motor) and the Hoehn and Yahr scale (HY). All the patients underwent magnetic resonance imaging (MRI) with a 3.0-T system. LA was rated using the semiquantitative visual rating system proposed by scheltens et al. RESULTS: Both the PD-IC (2.43 ± 2.79) and PD-MCI (4.48 ± 4.33) groups showed significantly lower deep hyperintensities (DHs) scores than the PDD group (7.88 ± 6.69, P = 0.004 and 0.040, respectively), especially in frontal (1.09 ± 1.31; 1.83 ± 1.90; 3.24 ± 2.64, P < 0.05) and parietal areas (0.09 ± 0.29; 0.65 ± 1.03; 1.53 ± 2.32, P < 0.05). There were no significant differences in periventricular (1.57 ± 1.75; 2.52 ± 2.37; 3.24 ± 2.64, P > 0.05), basal ganglia (0.09 ± 0.42; 0.30 ± 0.77; 0.53 ± 1.33, P > 0.05) and infratentorial white matter hyperintensities scores (--; 0.13 ± 0.63; 0.18 ± 0.73, P > 0.05) among three groups. The DHs showed a significant correlation with age (P = 0.003), MMSE (P = 0.009), verbal fluency test (P = 0.009), orientation (P = 0.047) and executive function (P = 0.027) in CAMCOG-C. The multiple regression analysis showed that the MMSE scores were associated significantly with education (P < 0.001, ß = 0.600), DHs (P = 0.001, ß = -0.678) and HY (P = 0.035, ß = -0.480). DHs were the most significantly associated with MMSE scores. CONCLUSION: There was a significant correlation between DHs and multiple domain cognitive impairment in PD, especially in executive function. DHs, which were the most significantly variable associated with MMSE scores, may contribute to cognitive impairment in PD.

[Vascular risk factors and cognitive impairment of patients with magnetic resonance imaging-defined subcortical ischemic depression].

OBJECTIVE: To explore the vascular risk factors and neuropsychological profiles of patients with magnetic resonance imaging (MRI)-defined subcortical ischemic depression (SID). METHODS: Thirty-five SID patients, 37 patients with subcortical ischemic vascular disease without depression (SIVD) and 40 normal controls (NC) participated in this case-control study. The SID and SIVD groups were recruited from the Department of Neurology, First Affiliated Hospital, Anhui Medical University between October 2010 and September 2011. And the NC group came from Medical Center, First Affiliated Hospital of Anhui Medical University over the same period. The vascular risk factors of all participants were assessed by the Framingham scale. Depression symptoms were assessed by the geriatric depression scale (GDS) and Hamilton depression scale (HAMD) while cognitive functions by the mini-mental state examination (MMSE) and Montreal cognitive clock drawing task (CDT). RESULTS: Both the SID (12.3 ± 4.7) and SIVD (13.5 ± 3.4) groups showed significantly higher Framingham scores than the NC group (8.6 ± 1.8) (F = 20.850, P = 0.000). Compared with the NC group (28.3 ± 1.8; 23.1 ± 3.4; 8.0 ± 1.8), the MMSE, MoCA and CDT scores were significantly lower in the SID (26.7 ± 2.5; 20.0 ± 4.0; 2.0 ± 2.7) and SIVD (26.8 ± 1.9; 20.7 ± 3.0; 6.1 ± 2.1) groups (P < 0.05). There were significantly lower CAMCOG-C scores in the SID (82.7 ± 9.0) and SIVD (86.2 ± 6.9) groups versus the NC group (92.3 ± 6.2) (P < 0.05) and similarly in the SID group versus the SIVD group (P < 0.05). Among the subtests of CAMCOG-C, the orientation, language and memory scores in both SID (9.0 ± 1.4; 24.7 ± 4.0; 19.5 ± 3.4) and SIVD (9.4 ± 0.9; 25.5 ± 2.1; 20.3 ± 2.3) groups were significantly lower than those in the NC group (9.9 ± 0.4; 27.4 ± 1.9; 22.1 ± 2.4) (P < 0.05); the praxis scores in the SID group (9.4 ± 1.9) were significantly lower than those in the NC group (10.4 ± 1.5) (P < 0.05). And the thinking and perception scores in the SID group (5.4 ± 1.7; 6.3 ± 1.4) were significantly lower than those in the SIVD (6.2 ± 1.3; 7.0 ± 1.4) and NC (6.6 ± 1.3; 7.2 ± 1.4) groups. In the SID group, no significant correlation was found between the Framingham scores and neuropsychological assessments. There were a negative correlation between the GDS, HAMD scores and MMSE, MoCA, CAMCOG-C and CDT scores (P < 0.05). CONCLUSION: The SID patients demonstrate multiple neuropsychological deficits, especially in thinking, perception and praxis. No significant correlation between vascular risk factors and depression symptoms is found in SID patients.

[Blood biochemical indices of female red-crowned crane (Grus japonensis) in Zhalong Nature Reserve of Heilongjiang Province, Northeast China].

From November 2004 to October 2005, twenty blood biochemical indices, i. e., total protein, serum albumin, serum globulin, total bilirubin, direct bilirubin, indirect bilirubin, blood glucose, serum urea nitrogen, serum creatine, total cholesterol, triglyceride, aspartate amino transferase, alanine amino transferase, alkaline phosphatase, serum creatine kinase, hydroxybutyrate dehydrogenase, lactic dehydrogenase, serum calcium ion, inorganic phosphate, and magnesium ion, of 10 female Grus japonensis adults in their wintering, reproduction, and migration periods in Zhalong Nature Reserve were analyzed by automatic biochemical analyzer. Significant differences (P < 0.05 or P < 0.01) were observed in the test indices except serum total protein, serum globulin, and blood glucose among the three life periods, which suggested that the serum total protein, serum globulin, and blood glucose could be used as the reliable references of blood biochemical indices of female G. japonensis, while the year-round dynamics of the other 17 indices reflected the physiological characteristics and ecological adaptability of female G. japonensis in its different periods in one year. When using these 17 indices as the references of the blood biochemical indices of female G. japonensis, the physiological period should be considered.

[Development of a DNA biochip for detection of known mtDNA mutations associated with MELAS and MERRF syndromes.].

We developed an oligonucleotide biochip for synchronous multiplex detection of 31 known mitochondrial DNA mutations associated with MELAS (Mitochondrial encephalomyopathy with lactic acidosis and stroke-like episodes) and MERRF (Myoclonic epilepsy with ragged red fibers). Allele-specific oligonucleotide probes were covalently immobilized on aldehyde modified glass slides, and then hybridized with Cy5-labled DNA fragments amplified from sample DNAs by a multiplex asymmetric PCR (MAP) method. Five patients with MELAS, 5 patients with MERRF and 20 healthy controls were investigated using the oligonucleotide biochip. The results showed that all the cases with MELAS had an A3243G mutation in the MT-TL1 gene. In the MERRF group, 4 cases were found to be an A8344G mutation and 1 case was a T8356C mutation, and both mutations were in the MT-TK gene. In the healthy controls, none of the 31 related mutations was found. The results of the DNA biochip were consistent with those by DNA sequencing. Clearly, the DNA biochip combined with MAP method would become a valuable tool in multiplex detecting of the point mutations in mtDNA leading to MELAS and/or MERRF syndrome. Moreover, this biochip format could be modified to extend to the screening scope of SNPs for any other human mitochondrial diseases.

[Construction and characteristics analysis of somatic cell lines from common carp Cyprinus carpio].

Epithelia of rostral side and fins from common carp were taken to culture in vitro. As a result, cells of tail fins were successfully passed from generation to generation, and constructed cell lines. During the culture procedure, the passage cells were observed and their biological characteristics were analyzed, which consist of morphologic analysis, drawing of growth curve, detection of producing rate for clones, insight of adaptation to temperatures, calculation of chromosomes and investigation for thaw of frozen cells. According to the observation results, we found that (1) there is an increase trend of fibroblast. epithelia of rostral side and tail fin from common carp are the best materials for culturing cells; (2) the optimum culturing temperature is 27 degrees C; (3) there isn't any change in cell chromosomes, which still are normal diploid. However, with, the generations increasing, the mode of chromosome has an inclination of decrease in normal ranges. We inferred the donor cells from 4-10 generations are the best choice for nuclear transplanted experiments in order to guarantee their genetic stabilities and survival rates after transplanting the nuclei. And, the cells will grow extremely well when cultured for 48-72h at 27 degrees C. We suggest the temperature should be descended little by little involving in preserving cells with DMSO. So far, the cells were passed 30th generations and part of them was preserved into liquid nitrogen.