冻结保存液对软枣猕猴桃腋芽冻结保存的影响(英文)

于12月取材的软枣猕猴桃侧芽通过酒精和次氯酸钠灭菌以后,以此为试材探讨了冻结保存液的组成、温度和浸泡时间对其冻结保存的影响。结果表明:最适的DMSO的浓度为15％,最适的浸泡温度为0℃。在侧芽预冷到-30℃后,再投入到液氮中,解冻后的生存率在含有甘油8 mol/L和11.5 mol/L区最高。用甘油预处理的实验结果表明:在处理浓度较高时可以获得较高的生存率。然而,在甘油预浸后,再用冻结保存液处理的最佳时间为小于1h。     

Effects of aging on sperm maturation and fertility in mice

AIM: To investigate the effects of aging on sperm maturation and fertility in mice. METHODS: Sperm of caput epididymides and cauda epididymides were obtained from Kunming mice aged 6 months (n=15, as control) and 18 months (n=15). Sperm parameters including sperm density, viability, motility and normal morphological rate were recorded. Sperm of cauda epididymides was observed by transmission electron microscope. The fertility potential and embryo developmental competence were performed by in vitro fertilization and embryo culture. RESULTS: Sperm motility, density and normal morphological rate in aged mice were lower than those in control (P<0.05). The fertilization rate and embryo developmental rate of aged group were significantly lower than those in control (P<0.01). CONCLUSION: Aging influences spermatogenesis in testes and sperm maturation in epididymides. Mouse would be served as a good model for male reproductive aging research.     

Inhibition of Rac1 protects cardiac functions in STZ-induced diabetic cardiomyopathy through decreasing pho-p38 MAPK

AIM: To investigate the effects of Rac1 inhibition on the size of cardiomyocytes and left ventricular functions in diabetic cardiomyopathy. METHODS: Type 1 diabetes was induced in 8-week old C57 mice by streptozotocin (STZ, ip injection). Diabetic mice were treated with NSC23766, a specific inhibitor of Rac1 (STZ+NSC group, n=15) or without treatment (STZ group, n=15). Nondiabetic mice were used to serve as empty control (Con group, n=10) or NSC23766 control (NSC group, n=10). The survival rate, LVHW/BW and left ventricular functions were detected at the end of 8 weeks after induction of diabetes. The expression of ANP, BNP, β-MHC and pho-p38 MAPK in the cardiac tissues were analyzed by real-time RT-PCR and Western blotting, respectively. Hematoxylin and Eosin staining (HE) was used to measure the cell size in the cardiac tissues. RESULTS: The functions of left ventricle were significantly impaired in the diabetic animals with decreased survival rate and increased LVHW/BW, which was accompanied by significant increases in ANP, BNP and β-MHC, and elevated pho-p38 MAPK expression in diabetic hearts. The increased survival rate, improved left ventricular functions and decreased LVHW/BW were observed in the diabetic animals treated with NSC. The mRNA expression of ANP, BNP, β-MHC and pho-p38 MAPK was significantly attenuated in the diabetic hearts by NSC treatment. The size of the cardiomyocytes, which increased in diabetic hearts, was decreased in the NSC-treated diabetic cardiac tissue. CONCLUSION: Rac1 inhibition protects left ventricular functions and attenuates hypertrophy, which is associated with the decrease in pho-p38 MAPK expression in diabetic heart. These data suggest that inhibition of Rac1 might be beneficial to diabetic cardiomyopathy.     

怀地黄玻璃化和包埋玻璃化法超低温保存

比较了玻璃化与包埋玻璃化超低温保存怀地黄（Rehmannia glutinosa(Gaertn.) Libosch）‘85-5’带芽茎段的效果。结果表明：4 ℃低温锻炼5 d,在添加二甲基亚砜（DMSO）和乙酰胺的预培养基中预培养3 d,60 % PVS₂室温装载25 min,PVS₂ 0 ℃脱水30 min时,玻璃化和包埋玻璃化两种保存方法均达到本试验条件下最佳结果;两种方法相比,玻璃化法的存活率和再生率均较包埋玻璃化法高,前者的存活率（88.92 %）和再生率（64.29 %）分别是后者的1.38倍和2.41倍。可见,采用超低温保存怀地黄种质资源是可行的,且玻璃化法优于包埋玻璃化法。     

Cryopreservation of coriander (Coriandrum sativum L.) somatic embryos using sucrose preculture and air desiccation

An efficient protocol for cryopreservation of somatic embryos of Coriandrum sativum, an important spice and medicinal herb, was developed. The successful cryopreservation procedure utilized embryo clumps (ECs) comprised of 3–4 somatic embryos at the globular or heart-shape stage. These ECs were precultured for 3 days on medium supplemented with 100 g/L sucrose, desiccated under the current of sterile air for 100 min, then sealed in cryovials and plunged directly into liquid nitrogen. Preliminary incubation on sucrose-enriched medium (100 g/L) improved both desiccation- and cryo-tolerance of ECs compared to medium with normal sucrose content (30 g/L) and enhanced embryo formation after cryopreservation. The regrowth after cryopreservation and average number of new embryos developed from cryopreserved ECs were retained at the level of the untreated control (98% and 13 embryos per clump, respectively). Both normal and abnormal plants were produced from control and cryopreserved cultures, indicating that appearance of abnormalities was not related to cryopreservation. The regenerants with normal phenotype showed the same peaks of relative DNA content regardless of cryopreservation. The results suggest that simple desiccation method is effective for cryopreservation of coriander somatic embryos with subsequent regeneration.     

Association between asthenospermia and mtDNA mutations in ND3 and ND4L genes

AIM: To explore the molecular mechanism of asthenospermia(AST) by preliminary screening of nucleotide sequences from the ND3 and ND4L genes of mitochondrial DNA(mtDNA). METHODS: Samples from 50 AST patients and 42 age-matched normal controls were collected according to the WHO criteria. Density gradient centrifugation was applied to separate spermatozoa with different vigor. The ND3 and ND4L genes of mtDNA were amplified and sequenced directly from the extracted genomic DNA from AST patients and normal controls. The sequences were compared with revised Cambridge Reference Sequence(rCRS) to analyze the variants. RESULTS: A total of 22 nucleotide variations were found in ND3 and ND4L genes of mtDNA in asthenospermia group and control group. G10320A, A10398G and T10609C were missense mutations, while A10157G and A10313C were the reported for the first time in this study. Haplotype N in patients with AST(33/50) was higher than that in control group(14/42, P<0.05), and haplotype R9 in patients with AST(15/50) was also higher than that in control group(4/42, P<0.05) through genetic testing of ND3 gene. Rates of sperm progressive motility of haplotype F1, F2 and R9 were significantly lower than those of haplotype M and M rest. Two haplotype differences, haplotype M and N, were found in the same AST patient’s spermatozoas which had different vigor. Haplotype M had stronger vigor, while haplotype N had lower vigor. By sequencing ND3 gene of mtDNA from 50 AST patients, we detected G10310A heteroplasmic mutation in 2 specimens of asthenospermia with poor and moderate motility spermatozoa, respectively. No mutation occurred in good motility spermatozoa. CONCLUSION: Haplotype of mitochondrial may have some correlation with sperm motility. The nt10398G-10400T polymorphisms may have benefit for sperm motility, whereas the mutation in nt10310A may impair sperm motility.     

香蕉种质超低温保存技术研究进展

对于香蕉这种营养繁殖的植物,超低温保存是长期有效地保存其种质的一种有效方法。主要综述了香蕉胚性悬浮细胞系、合子胚和茎尖分生组织的超低温保存技术方面近十几年来的研究进展。总结了影响香蕉超低温保存效果的几个主要因素,主要包括材料、预处理、装载处理以及冰冻保护剂。并分析了当前应用在香蕉分生组织上的4种超低温保存方法的优缺点,分别为:分生组织团简单冻存法、分生组织团玻璃化法、单个分生组织玻璃化法、滴冻玻璃化法,认为滴冻玻璃化法效果最好。     

蔬菜可溶性固形物含量与冰点温度的关系

利用HP34970A数据采集仪和计算机联用,测定了13种蔬菜活组织的冰点温度,利用PR-101型折光仪测定了蔬菜可溶性固形物含量。结果表明,蔬菜可溶性固形物含量(X)和冰点温度(Y)是高度负相关(r=-0.9007),其回归方程为蔬菜组织冰点Y=-0.2419X-0.3358。13种蔬菜活组织均有典型的冰点曲线。     

铁皮石斛种子、原球茎和类原球茎体的超低温保存研究

对珍稀濒危植物铁皮石斛的种子、原球茎和类原球茎体进行了液氮超低温保存研究。把４个月龄的种子脱水至含水量为１２％～１９％,能获得较高的冻后存活率（９５％）和较快的恢复生长速率。种子在０．５ｍｏｌ／Ｌ脱落酸培养基上萌发３周所形成的原球茎,用玻璃化保护剂ＰＶＳ２处理１５ｍｉｎ,其冻后存活率可达８８％。将生长旺盛的暗培养类原球茎体的含水量在６～７ｄ内干燥至３０％±２％,其冻后存活率可达４８％～８０％。     

番木瓜茎尖超低温保存过程中的细胞超微结构

 采用透射电子显微镜对番木瓜(Carica papaya L. ) 茎尖超低温保存过程中细胞超微结构变化进行观察。结果表明: 细胞在经过预培养、60% PVS₂ 预处理和PVS₂ 脱水处理, 细胞质壁分离程度逐渐加重,细胞的抗冻力增加。细胞严重伤害主要发生在液氮保存的降温及解冻的过程中, 一部分细胞的细胞壁、细胞膜以及细胞核膜均有不可逆的损伤, 可能是导致部分细胞致死的原因之一。也有部分细胞结构完整, 虽然细胞结构发生了变化, 但是程度不深, 是可逆的伤害, 在恢复培养3 d时可自动修复, 然后再生出植株。     

金太阳杏花粉超低温保存方法研究

为探讨杏花粉种质保存的方法,进而为花粉培养、遗传转化和杂交育种提供材料,以杏品种金太阳的新鲜成熟花粉为试材,采用干燥法和玻璃化法2种超低温保存技术,对花粉干燥时间、解冻方式和贮藏时间等影响保存后花粉萌发率的有关因素进行研究。结果表明,干燥时间对金太阳杏花粉超低温保存后的萌发率有显著影响,其中干燥法宜在4℃下硅胶干燥8h、玻璃化法宜在(20±1)℃下以玻璃化液PVS2(体积分数15%二甲基亚枫+15%乙二醇+30%甘油+0.4mol/L蔗糖)处理60min;干燥法保存后分别以4℃2h、20℃30min和40℃70s等3种方式化冻,花粉萌发率无明显差别,而对玻璃化法则以40℃70s化冻后的花粉萌发率最高;以2种方法分别保存1、10、30、60d后花粉萌发率无显著变化。     

Effect of Siduqing decoction,a Chinese medicine,on survival rate and multiple organ dysfunction in mice challenged with LPS

AIM: To investigate the effect of siduqing decoction, a Chinese medicine, on survival rate and multiple organ dysfunction in mice challenged with LPS. METHODS: Mice were administered intragastrically with Siduqing decoction or distilled water (0.2 ml/10 g) twice a day for 3 days, two hours after Chinese herbal medicine treatment on day 3, LPS or normal saline was injected intraperitoneally, and survival rates in each group were recorded at 12-h intervals. In another experiment, mice were sacrificed at 12 h after LPS, lung, liver, kidney and small intestine were collected and processed for the H & E staining. In addition, Blood was collected at 10 h after LPS injection for determining alanine aminotransferase (ALT) activity, blood urea nitrogen (BUN) and creatinine (Cr) contents. RESULTS: At 96 h after LPS injection, the survival rate (27%, n=34) was lower in LPS group than Siduqing treatment group (65%, n=31, P<0.05). ALT activity, BUN and Cr contents in serum were higher in LPS group than control group, Siduqing treatment significantly attenuated a increase in ALT activity, BUN and Cr content in serum induced by LPS. Histological examination showed inflammatory injury in the lung and intestine, hemorrhage in the lung and kidney, degeneration, necrosis in the liver and kidney, while Siduqing treatment attenuated pathological changes induced by LPS. CONCLUSION: These data indicate that Siduqing has a protective effect against LPS-induced multiple organ injury and increases survival rate in mice challenged with LPS.     

Evaluation of antisperm antibody in infertile women with polycystic ovary syndrome

AIM: To investigate the level of circulatory antisperm antibody (ASA) in infertile women with polycystic ovary syndrome (PCOS). METHODS: A total of 127 infertile women with PCOS (26~35 years old) were divided into 2 groups according to the body mass index (BMI):obese group (BMI ≥ 25 kg/m², n=42) and non-obese group (18 kg/m² < BMI < 25 kg/m², n=85). The infertile women aged 24~36 years of normal weight without PCOS (n=90) were chosen as controls (control group; 18 kg/m² < BMI < 25 kg/m²). The serum levels of ASA-IgG and ASA-IgA were screened by the indirect immunobead test according to the WHO laboratory manual. The 50% or more of the motile sperm attached to one or more immunobeads were regarded as clinical positivity according to the WHO criteria. The 20%~49% motile sperm that had adherent particles were deemed to be weakly positive. Less than 20% was negative. RESULTS: No case in obese group and non-obese group showed the positive level of ASA-IgG. Two cases in control group were detected to be ASA-IgG positive (2.2%). One case in obese group (2.4%), 2 cases in non-obese group (2.2%) and 2 cases (2.2%) in control group were detected to be ASA-IgG weakly positive, and no difference was found in the weakly-positive incidence among these 3 groups. ASA-IgA was not detected in all the cases. CONCLUSION: The circulatory ASA is not associated with the pathogenesis of infertile women with PCOS. The detection of ASA still needs to be routinely performed for infertile women.     

香菇单核菌株菌丝生长特性分析

为研究不同品种香菇(Lentinus edodes)单核菌株菌丝生长的差异性,选取13个香菇品种,采用单孢分离法获得其单核菌株。培养并统计其菌丝生长速率、菌落长势评分、菌落形态类型等指标的分布频率及低温保藏对单核菌株菌丝生长的影响。结果表明,不同品种的单核菌株菌丝生长速率差异显著且分布状况不同,其中,0912单核菌株的菌丝生长速率最快为2.02 mm·d-1,L-02单核菌株的菌丝生长速率最慢为0.77 mm·d-1;不同香菇品种的菌落类型分布频率差异很大,菌落长势评分分布符合正态分布;长时间低温保藏可导致单核菌株菌丝萌动期延长,菌丝生长速率下降;同一品种内的单核菌株菌丝生长特性差异显著。     

An encapsulation–dehydration protocol for cryopreservation of the azalea cultivar ‘Nordlicht’ (Rhododendron simsii Planch.)

Azalea is important for Flanders (Belgium). An in vivo breeding collection, with more than 600 accessions, is available at the Department of Plant Genetics and Breeding (CLO, Melle, Belgium). The aim of our research is to develop a cryopreservation protocol for this collection, one of the most important in the world. With ‘Nordlicht’ as test cultivar, we developed an encapsulation–dehydration protocol. Optimal survival was observed after preculturing shoot tips in liquid culture medium supplemented with 0.3 + 0.45 + 0.6 M sucrose (24 h in each solution). After preculture, shoot tips were encapsulated in alginate beads (1.62 M glycerol + 0.32 M sucrose) and dehydrated to a low WC (38.6%) prior to freezing (direct immersion in LN). Detoxification in 1 M sucrose (2 h) appeared to be essential to achieve acceptable regrowth after cryopreservation. After thawing approximately 40% of the cryopreserved shoot tips survived.     

Long-term effects of different drugs on contralateral testis with unilateral testicular torsion in prepubertal rats

AIM: To observe the long-term effects of taurine, Danxuetong (Salvia miltiorrhiza combined with Xueshuantong) and breviscapine on contralateral testis with unilateral testicular torsion in prepubertal rats, and to compare their protective effect on testis with ischemia reperfusion injury. METHODS: Four-week-old healthy male SD rats (n=64) were randomly divided into 8 groups: sham operation group, saline group, single-dose and successive taurine groups, single-dose and successive Danxuetong groups, and single-dose and successive breviscapine groups. The animal model of left testicular torsion reduction (720° for 2 h) was established. After 6 weeks of surgery, the right testes and epididymides from the rats were collected immediately to detect the total antioxidant capacity (T-AOC), the activity of superoxide dismutase (SOD), the activity of nitric oxide synthase (NOS) and the content of malondialdehyde (MDA). The sperm deformity, sperm viability and sperm concentration were measured, and the pathological examination of the testicular tissues was performed. RESULTS: Compared with saline group, the activity of SOD, T-AOC, sperm viability and sperm concentration were increased, while MDA content and sperm deformity were decreased in 3 successive groups. SOD activity and sperm viability (except single-dose breviscapine group) were increased, but MDA content and sperm deformity were decreased in 3 single-dose groups. T-AOC activity and sperm concentration were increased in single-dose taurine group. The saline group showed interstitial edema and degeneration of seminiferous tubules, and the rest groups showed a significant improvement in histological morphology. CONCLUSION: The contralateral testis in prepubertal rats was damaged after unilateral testicular torsion, and taurine, Danxuetong and breviscapine have long-term protective effect on the tissues under ischemia-reperfusion injury. The protective effect of Danxuetong is better than breviscapine, and taurine is the best of them. The effect of single-dose group is better than the successive application.     

Effect of ligustrazine on protein kinase C signaling pathway in human peripheral blood lymphocytes

AIM:To investigate whether Ligustrazine(LTZ) has an effect on the changes of protein kinase C(PKC) signaling pathway induced by inflammatory mediators involved in asthma in normal human peripheral blood lymphocytes (PBL).METHODS:10 mL peripheral venous blood was obtained from each of 63 health humans and treated as follows. The activities of PKC from cytosolic and membrane fractions in PBL were measured by -ATP-catalyzing assay, after PBL had been isolated and performed by following processes: (1) First: three groups treated with 5 g/L LTZ(n=6) or 5 μmol/L Ro31-8220 (n=6); Paired untreated PBL served as control of this group, as well as the negative controls of the following groups(n=6); (2)Second : three groups treated with 100 nmol/L Methacholine (Mch, n=5), 5 g/L LTZ+100nmol/L Mch(n=5)or 5 μmol/L Ro31-8220(a PKC inhibitor)+100 nmol/L Mch(n=5); (3)Third: three groups treated with 100 nmol/L histamine, 5 g/L LTZ+100 nmol/L histamine(n=5) or 5 μmol/L Ro31-8220+100nmol/L histamine(n=5); (4)Fourth: three groups treated respectively with 100nmol/L PMA(a PKC activator, n=5), 5 g/L LTZ+100nmol/L PMA(n=5) or 5 μmol/L Ro31-8220+100nmol/L PMA(n=5).RESULTS:(1)LTZ had no effect on the activities of PKC in inactive PBL in normal humans; (2) Methacholine or histamine resulted in an increase in membrane PKC activity of normal human PBL, which was partly suppressed by LTZ (all P＜0.05); (3) PMA caused an increase in membrane PKC activity of normal human PBL, which was partly decreased by LTZ (all P＜0.05).CONCLUSION:LTZ has an inhibitory effect on activation of PKC signaling pathway in PBL in normal humans induced by some inflammatory mediators involved in asthma, which may be one of the mechanisms that LTZ plays a role in the prevention and therapy of asthma.     

苹果离体茎尖超低温保存方法的比较

 以苹果离体茎尖为试材，对4种超低温保存方法(两步降温法、玻璃化法、包埋干燥法和小滴冰冻法)从操作程序、存活率、再生率及恢复生长速度等方面进行比较，确定包埋干燥法为最佳保存方法。     

Protective effect of atrial natriuretic peptide on alveolar type II cells

AIM: To study the protective effect of atrial natriuretic peptide (ANP) on alveolar type II cells (AT-Ⅱ) damaged by lipopolysaccharide (LPS).METHODS: AT-Ⅱ were placed in a 6 well cell culture cluster (0.5×106 cells/cm2) and divided into 3 groups: (1) control group (n=6), the medium consisted of RPMI-1640 without FBS. (2) LPS group (n=6), the medium consisted of RPMI-1640 without FBS supplemented with LPS (1 mg/L). (3) ANP group (n=6), the medium consisted of RPMI-1640 without FBS supplemented with LPS (1 mg/L) and ANP (10-8, 10-7, 10-6 mol/L). After 4, 12 and 24 h, the cell culture mediums of control group, LPS group and ANP (10-7 mol/L) group were collected, and those of the ANP (10-6, 10-8 mol/L) group were collected after 12 h. Alkaline phosphatase(AKP), lactate dehydrogenase(LDH), malondialdehyde(MDA), total phospholipids (TPL) and surface tension (ST) in the medium of every group were examined. RESULTS: AT-Ⅱ were characterized by AKP staining. The contents of LDH, AKP and MDA in the medium of every ANP group were lower than those in the corresponding LPS group. The TPL content in the medium of every ANP group was higher than that in the corresponding LPS group, and the change of ST of the medium was opposite to that of TPL. The effect at 12 h was the most significant, for example, at 12 h, the activities of AKP in the mediums were: control (43.5±10.4) U/L, LPS (98.1±16.4) U/L, LPS+ANP (10-6) (46.4±10.5) U/L, LPS+ANP(10-7) (60.7±9.5) U/L, LPS+ANP(10-8) (91.3±13.9) U/L.CONCLUSION: ANP protects the AT-Ⅱ from being damaged by LPS and promotes the secretion of pulmonary surfactants.     

菌种冷冻保藏新技术研究初报

本研究以普通冰箱冷冻室对麦粒菌种进行冷冻保藏，其中用生石灰干燥的参试菌种成活率达到100％，干燥方法和冷冻方法均简便易行，因此具有普遍的实用意义。