Direct Regeneration of Plants Derived from in vitro Cultured Shoot Tips and Leaves of Poplar （Populus×euramericana ＇Neva＇）

The purpose of the present study was to establish a regeneration procedure for Populus × euramericana ＇Neva＇ by using in vitro shoots tips and leaves. For sterilization, 0.1% （w/v） mercuric chloride （HgCl2） solution for 8 to 10 min was the optimal treatment for this poplar cultivation. The effects of benzyladenine （BA） and α-naphthaleneacetic acid （NAA） added to Murashige and Skoog （MS） medium were tested on organogenesis. The highest regeneration rate and numbers of shoots/explant from shoot tips （96.7%, 9.8） and leaves （90.0%, 8.7） were obtained on the half-strength MS medium supplemented with 0.5 mg/L BA and 0.1 mg/L NAA. The optimal medium for in vitro rooting of shoots was on a half-strength MS medium containing 1 mg/L indolebutyric acid （IBA） with the highest rooting frequency （93.3%） and numbers of roots/explant （8.2）. For acclimatization, in vitro rooted plantlets were transferred to plastic cups containing vermiculite and peat （1： 1）. After acclimatization, transplanted plantlets grew well in a shade house. Therefore, we believe that this efficient plant regeneration protocol especially by leaf explants is very important for in vitro clonal propagation of Populus×euramericana ＇Neva＇.

IGF-1 mRNA expression of adult rat thyroid cell cultured in vitro

Objective: To investigate the law of age-related changes of insulin-like growth factor-1 (IGF-1) expression of rat thyroid cells cultured in vitro. Methods: Rat thyroid of different age (10, 45, 65, 100, 150 weeks) was isolated and thyrocytes cultured. Total RNA was extracted in different rat age group when thyroid cells had been cultured for two weeks. mRNA IGF-1 expression was measured with reverse-transcription polymerase chain reaction( RT-PCR) in each group and compared. Results: Quantity of total RNA in thyroid cells decreased with ageing when the rat thyroid cells had been cultured for 2 weeks. There is significant difference among groups (P<0.05). Expression of IGF-1 mRNA could be detected in thyroid cells of different age cultured in vitro. Quantity of IGF-1 mRNA expression by RT-PCR analysis increased from 10 to 45 weeks old, and then decreased with ageing. Conclusion: Rat thyroid cells from different age cultured in vitro can express IGF-1 mRNA. Quantity of total RNA in thyroid cells cultured in vitro decreased with aging. IGF-1 mRNA expression was correlated to age (r=0.401, P<0.05).

Effect of polygonatum polysaccharide on the hypoxia-induced apoptosis and necrosis in in vitro cultured cerebral cortical neurons from neonatal rats

BACKGROUND： Cardiocerebrovascular diseases induced cerebral circulation insufficiency and senile vascular dementia can result in ischemic/hypoxic apoptosis of central neurons, which we should pay more attention to and prevent and treat as early as possible. Traditional Chinese medicine possesses the unique advantage in this field. Polygonatum, a Chinese herb for invigorating qi, may play a role against the hypoxic apoptosis of brain neurons. OBJECTIVE ： To observe the protective effect of polygonatum polysaccharide on hypoxia-induced apoptosis and necrosis in cerebral cortical neurons cultured in vitro. DESIGN： A comparative experiment.SETTING： Laboratory of Cell Biology, Institute of Basic Medical Sciences, Jiangxi Provincial Academy of Traditional Chinese Medicine. MATERIALS： The experiment was carried out in the Laboratory of Cell Biology, Institute of Basic Medical Sciences, Jiangxi Provincial Academy of Traditional Chinese Medicine from November 2003 to April 2005. Totally 218 Wistar rats （male or female） of clean degree within 24 hours after birth were purchased from the animal center of Jiangxi Medical College （certification number was 021-97-03）. METHODS：① Preparation of cerebral cortical neurons of rats： The cerebral cortical tissues were isolated from the Wistar rats within 24 hours after birth, and prepared to single cell suspension, and the cerebral cortical neurons of neonatal rats were in vitro cultured in serum free medium with Neurobasal plus B27 Supplement. ② Observation on the non-toxic dosage of polygonatum polysaccharide on neurons： After the neurons were cultured for 4 days, polygonatum polysaccharide of different dosages （1-20 g/L） was added for continuous culture for 48 hours, the toxicity and non-toxic dosage of polygonatum polysaccharide on neurons were observed and detected with trypan blue staining. ③Grouping： After hypoxia/reoxygenation, the cultured neurons were divided into normal control group, positive apoptotic group and polygonatum polysaccharide group. In the normal control group, the neurons were cultured at 37℃ in CO2 with the volume fraction of 0.05 under saturated humidity for 6 days. In the apoptotic positive group, the neurons were cultured with hypoxia for 12 hours after 4-day culture, and followed by reoxygenation for 48 hours. In the polygonatum polysaccharide group, polygonatum polysaccharide with the terminal concentration of 0.5, 1 and 1.5 g/L was added to some neurons at 10 hours before the hypoxia culture, and then the neurons were cultured with hypoxia for 12 hours, followed by reoxygenation for 48 hours; polygonatum polysaccharide with the terminal concentration of 0.5, 1 and 1.5 g/L was added to the other neurons at 12 hours after hypoxia followed by reoxygenation for 48 hours.④ The Hoechst33342 fluorescence staining, Annexin V/PI flow cytometer, appearance of DNA agarose gel electrophoresis gradient strap and immunohistochemical staining were used to observe the expressions of Bcl-2, Bax and Caspase-3 apoptotic and anti-apoptotic proteins and the ratio of Bcl-2/Bax, and observe the effect of polygonatum polysaccharide against the hypoxic apoptosis of cerebral cortical neurons of neonatal rats. MAIN OUTCOME MEASURES： ① Toxicity and non-toxic dosage of polygonatum polysaccharide on neurons;② Apoptotic rate of neurons detected with Hoechst33342 fluorescence staining;③ Early apoptotic rate and necrotic rate of neurons detected with Annexin V/PI flow cytometer; ④DNA agarose gel electrophoresis ladder-like strap appeared or not;⑤ Expressions of Bcl-2, Bax and Caspase-3 apoptotic and anti-apoptotic proteins and the ratio of Bcl-2/Bax. RESULTS：① Polygonatum polysaccharide within 6 g/L had no cytotoxicity on the normal cultured cerebral cortical neurons （P 〉 0.05）. ②The apoptotic rates of neurons detected with Hoechst33342 fluorescence staining had significant differences between the polygonatum polysaccharide groups and positive apoptosis group added to neurons at 10 hours before the hypoxia culture [（13.00±4.52）%,（12.72±2.15）%, （11.80±1.18）%,（38.03±1.05）%, P 〈 0.01], and had no significant differences between the polygonatum polysaccharide groups and positive apoptosis group added to neurons at 12 hours after the hypoxia culture （36.77±1.45）%, （36.60±1.61）%, （36.37±2.02）%, （38.03±1.05）%, P 〉 0.05].③ Annexin V/PI flow cytometer detected that the anti-necrotic effect was enhanced with the increased concentration of polygonatum polysaccharide within 0.5-1.5 g/L （P 〈 0.01）. Polygonatum polysaccharide of 0.5-1.5 g/L added before hypoxia could significantly decrease the apoptotic rate of neurons induced by hypoxia/reoxygenation （P 〈 0.01）. ④ No DNA agarose gel electrophoresis ladder-like strap appeared in the groups with polygonatum polysaccharide of 0.5-1.5 g/L added at 10 hours before hypoxia;⑤ After Polygonatum polysaccharide of 0.5-1.5 g/L was added before hypoxia, the expression of Bcl-2 protein of hypoxic neurons was increased （P 〈 0.01）, and those of Bax protein and Caspase-3 protein were reduced （P 〈 0.01）, and the ratio of Bcl-2/Bax was increased （P 〈 0.01）. CONCLUSION： Polygonatum polysaccharide within 6 g/L has no cytotoxicity on the normal cultured cerebral cortical neurons. Polygonatum polysaccharide of 0.5-1.5 g/L added before hypoxia plays a role agains necrosis of neurons induced by hypoxia. Polygonatum polysaccharide of 0.5-1.5 g/L can significantly reduce the apoptosis of neurons induced by hypoxia through up-regulating the expression of Bcl-2 protein, down-regulating the expressions of Bax protein and Caspase-3 protein, and increasing the ratio of Bcl-2/Bax.

Quantitative detection and comparison of sulfate glycosaminoglycans content in extracellular matrix of in vitro cultured epiphyseal, articular and rib chondrocytes

Objective To establish a method for quantitative detection of the sulfate glycosaminoglycans ( GAG) content in extracellular matrix of in vitro cultured chondrocytes so as to evaluate the biological characteristics of epiphyseal, articular and rib chondrocytes. Methods Sulfate GAG content in extracellular matrix of three chondrocytes was measured by the modified dimethylmethylene blue (DMB) method. The changes of the toluidine blue (TB) stain of chondrocytes were observed by light microscope. Results Primary chondrocytes had the highest content of sulfate GAG in the extracellular matrix, ie, epiphyseal chondrocytes reached ( 70. 12 ± 7. 72 )μg/cm2, articular chondrocytes (92.00 ± 10.15) μg/cm2 and rib chondrocytes (80.61 ± 11. 40) μg/cm2, respectively. On the third pasage chondrocytes, epiphyceal chondrocytes decreased to (53.27 ± 9. 50 ) μg/cm2, articular chondrocytes to (63.88 ± 11.92) μg/cm2 and rib chondrocytes to (58.94 ±8.21) μg/cm2, respectively. The change of TB in every passage

Effects of Hormone Factors on the in vitro Culture Flowering Induction of Dendrobium officinate Kimura et Migo

[Objective]The aim was to select the suitable hormone factors for flowering induction in vitro culture of Dendrobium officinate Kimura et Migo.[Method]The test-tube plantlets from the stems of Dendrobium officinate Kimura et Migo were used as the experimental materials and MS medium as the basic medium.Comparative tests have been done between single-factor hormone treatments（different concentrations of PP333 or TDZ） and multi-factor hormone treatments（different combinations of PP333,TDZ,6-BA and NAA） to research the effects of hormone factors on the flowering induction of the plantlets.[Result]Among the single-factor hormone treatments,the suitable concentration and the rate of flower buds formation of PP333 treatment were 0.2 mg/L and 8.5%,the that of TDZ treatment were 0.06 mg/L and 15.5%;the effects of multi-factor hormone treatments on the flowering induction were ordered as follow：（PP333 ＋ 6-BA ＋ NAA ＋ TDZ）〉 （PP333 ＋ 6-BA ＋ NAA）〉 （PP333 ＋ 6-BA） and（PP333 ＋ NAA） ;the most suitable treatment was PP333 0.3 mg/L ＋ 6-BA 0.5 mg/L ＋ NAA 0.5 mg/L ＋ TDZ 0.06 mg/L,the rate of flower bud formation and the rate of the blossomed flower were respectly reached to 80.4% and 90.3%.[Conclusion]PP333 and TDZ showed the important effect on the flowering induction in vitro culture of Dendrobium officinate Kimura et Migo.The effect of TDZ was better than that of PP333.It is much more conducive to the flower bud formation,when using appropriate concentration of TDZ combined with other hormones properly.

Rapid Propagation of Chirita ophiopogoides in Vitro

A method for in vitro culture and rapid propagation of Chirita ophio- pogoides was developed using leaves as explants in this study, The results indicat- ed that the medium MS＋6-BA 0.1 mg/L＋NAA 0.1 mg/L was suitable for bud induc- tion and seedling regeneration from leaves in primary culture. The media MS＋0.5 mg/L 6-BA＋0,1 mg/L NAA＋10% banana＋5% potato and MS＋0.5 mg/L 6-BA＋0.5 mg/L NAA＋2% banana were very suitable for callus multiplication and seedling hardening in subculture, and the proliferation coefficients were 7,9 and 5.6 per 60 d respec- tively. The optimal rooting medium was MS and the rooting rate was 100% on day 30 of culture. The rooted plantlets of C. ophiopogoides were transplanted in green- house with humus soil and 92.5% survived. Theoretically, using the rapid propaga- tion system, about 20 176 seedlings can be reproduced from a sterile plantlet in a year.

Culture Experiment in vitro of Eperythrozoon of Mustela lutreola

[Objective] The research aimed to make the culture experiment in vitro of eperythrozoon of Mustela lutreola. [Method] 30% newborn bovine serum or Mustela lutreola was added in RPMI-1640, DMEM, M-199 and HL culture solutions. Epery-throzoon of M. lutreola was cultured in vitro under the conditions of 37 ℃, 5% CO2. The culture solutions were replaced every 24 hours. Appropriate amount of healthy erythrocytes were supplemented to make subculture. [Result] The infection rate and infection intensity of eperythrozoon in RPMI-1640 and HL culture solutions were higher than that in DMEM and M-199 culture solutions. The infection rate and infec-tion intensity of eperythrozoon in RPMI-1640 and HL culture solutions with adding 30% serum of M. lutreola were higher than that with adding 30% newborn bovine serum. And the infection rate and infection intensity of eperythrozoon in RPMI-1640 culture solution were slightly higher than that in HL culture solution. The infection rate was over 80% after culturing 12 hours. 23 generations of subculture in vitro was made. [Conclusion] The research provided an effective approach for screening out the drugs that could effectively control eperythrozoon of M. lutreola, preparing diagnostic antigen and producing the relevant vaccine.

In vitro Culture of Unfertilized Ovary of Strawberry(Fragaria spp.)

[Objective] This study was to investigate the in vitro culture of unfertilized ovary of strawberry.[Methods] Employing single factor experiment,we investigated six key factors including genotype,extrinsic hormone,sucrose concentration,low temperature pretreatment,growth environment and development status,and illumination condition on induction of gynogenesis in vitro of unfertilized ovary,on the induction of gynogenesis in vitro of unfertilized ovary.[Results] The optimal conditions for in vitro culture of unfertilized ovary of strawberry were as follows：the primary flower buds cultured on bare land as explants,selection of appropriate genotype,2,4-D as external hormone,sucrose at the concentration of 6%,low temperature pretreatment for 48 hours and dark culture under alternated temperature.[Conclusion] The research provided reference for ploidy breeding in strawberry.

Effect of pre-culture on virus elimination from in vitro apple by thermotherapy coupled with shoot tip culture

We evaluated the role of pre-culture on survival rate of in vitro apple plants treated by thermotherapy. Two apple cultivars, Malusxdomestica cv. Pink Lady and Huafu, were used in the experiment and both have widely grown in China and infected with Apple chlorotic leafspot virus （ACLSV） and Apple stem grooving virus （ASGV）. Results in growth and virus titer of apple plants did not exhibit clear trends during five different periods of pre-culture. Whilst, pre-culture increased the survival rate of the two cultivars during thermotherapy. The survival rate of plants pre-cultured for 13 d （P-13d） was 14 and 51% higher than that of P-ld plants for Pink Lady and Huafu, respectively. Moreover, pre-culture positively influenced regeneration of Huafu plants. The average survival rate of plants regenerated from P-ld and P-4d was 20% lower than that regenerated from P-7d, P-10d, and P-13d. The efficiency of virus eradication was determined by reverse-transcription PCR with two primer pairs for each virus, and the detection results showed that pre-culture scarcely affected apple virus elimination. Despite the fact that the two viruses were hardly detected at 5 d of thermotherapy, no virus-free plants were found in the two cultivars of regenerated apple plantlets after 30-d treatment.

Echinococcus Granulosus: Suitable in vitro Protoscolices Culture Density

The present study is to determine the suitable protoscolices （PSCs） density for long-time culturing in vitro. The PSCs were divided into eight groups with different densities and the viability tests were carried out with 0.1% methylene blue staining. Then the infection ability of cultured PSCs was assessed by the mean cyst weight of mice inoculated intraperitoneally with PSCs after 8 months post-infection.

Studies on Electrical Activation of Porcine Oocytes Matured in vitro and Embryo Culture Systems

Conditions for electrical parthenogenetic activation of porcine oocytes matured in vitro and in vitro culture systems of porcine embryo were studied. The best results were achieved under the conditions of electrical field strength and the pulse duration at 130Vmm-1/80 us, with a blastocyst development rate of (20.12 ± 8.18) % (P > 0.05). No significant difference was found between treatments of multiple pulses and a single pulse (P > 0.05). Parthenogenetic embryos were cultured with different methods and air conditions for 7 days in vitro, blastocyst development rate of embryos with changed culture media [ (26.44 ± 8.35) % ] or changed media with 10% fetal bovine serum (FBS) [ (17.68 ± 5.39)% ] on the fifth day showing no significant difference from that of embryos without change of culture media [ (25.30 ± 7.55) % , P > 0.05 ], while cell numbers of blastocysts from embryos with changed culture media (15.78 + 5.46 and 14.55 ± 4.81) were significantly lower than number of blastocysts from embryos without change of culture media (18.01 ± 6.79, P<0.01). Blastocyst development rate and blastocyst cell number of embryos cultured in lower O2(5%CO2: 7%O2:88%N2) also showed no significant difference from those in high O2(5%CO2 in air) [(20.78 ± 8. 80)% and 17.0016.12 vs. (25.30 ± 7.55)% and 18.0116.79, P>0.05]. It is concluded that change of culture media with the same new one or changing over to media with 10% fetal bovine serum (FBS) on the fifth day and low O2 environment are not necessary for porcine embryos development.

Establishment of in vitro Rapid Propagation System for Ampelopsis grossedentata

[Objective] The research aimed to establish the fast and efficient rapid propagation system for Ampelopsis grossedentata by using the tissue culture method. [Method] Ampelopsis grossedentata stem with the bud was the material,and the influences of different sterilization methods,media and hormone combinations on the in vitro rapid propagation of Ampelopsis grossedentata were studied. [Result] The explant sterilization effect of 75% ethanol dipping 20 s ＋ 0.1% HgCl2 treating 8 min ＋ Tween-80 1-2 drops was better. The optimal medium for the axillary bud induction was B5 ＋ 1.00 mg/L BA ＋ 0.05 mg/L NAA. The optimal multiplication medium was MS/B5 ＋ 1.50 mg/L BA ＋ 0.05 mg/L NAA. 1/2MS ＋ 0.50 mg/L IBA was the best medium for the rooting. [Conclusion] The high frequency occurrence system of Ampelopsis grossedentata in vitro rapid propagation was established. It laid the technology basis for the callus regeneration system,genetic transformation and clonal mutation screening.

Toxicity Evaluation of in vitro Cultures of Freshwater Cyanobacterium Microcystis aeruginosa:Ⅰ.Hepatotoxic and Histopathological Effects in Rats

Laboratory cultures of freshwater cyanobacterium (blue-green alga) Microcystis aeruginosa PCC 7806 was cvaluated for its hepatotoxic effects in rats. The lyophilized cell extract injected intraperitoneally at 1 and 2 LD50 (15.8 and 31.6 mg/kg, respectively) produced significant increase in liver-specific enzymes viz. plasma alkaline phosphatase,γ-glutamyl transferase, lactate dehydrogenase with a concomitant decrease in hepatic glutamic pyruvic transaminase. A corresponding increase in liver body weight index and histopathological changes in liver (degeneration of hepatocytes, congestion and hemorrhage etc.) are indicative of a dose and time dependent hepatotoxic nature of the algal extract

Effect of Clenbuterol Hydrochloride on the in vitro Development of Mouse Embryo

To investigate the effect of clenbuterol hydrochloride on the in vitro development of both 1 cell and 2 cell mouse embryos. Methods The cultural systems of both 1 cell and 2 cell mouse embryo were used to determine the effect of clenbuterol hydrochloride at doses of 1 ng/mL, 3 ng/mL, and 10 ng/mL on developmental rates of mouse embryos. Results When 1 cell embryos cultured with 1 ng/mL of clenbuterol hydrochloride, developmental rates from the 4 cell stage to blastocyst stage were significantly lower than those in the control group (P<0.05), but on dosages of 3 ng/mL and 10 ng/mL, the inhibiting effects on embryo development were significantly increased (P<0.01). When 2 cell embryos cultured with 1 ng/mL of clenbuterol hydrochloride, obvious differences in developmental rates were not found between the 2 cell embryo group and the control (P>0.05). However, at levels of 3 ng/mL and 10 ng/mL, significant decrease of developmental rates in 2 cell embryos was observed from the 4 cell and from the 8 cell stage, respectively (P<0.05). Embryos cultured with clenbuterol hydrochloride appeared to have more granules, fragments and degeneration than those in the control. Conclusion Clenbuterol hydrochloride has a toxic effect on the mouse embryos, and the effect is in a dose dependent. 1 cell mouse embryos cultured with clenbuterol hydrochloride could be easily inhibited at 2 cell stage, but the effect of clenbuterol hydrochloride on development of the late 2 cell embryos would be reduced.

Studies on Single Cell Culture in vitro in Wheat——The variation of grain protein content and its fractions from regenerated plants

On the basis of previous studies dealing with the variation of major agronomic and yield characteristics of regenerated plants derived from single cell culture in vitro of common wheat (Triticum aestivum L.Cultivar NE 7742), the grain protein content and its fractions from regenerated plants with stable agronomic characteristics were studied from 1992 to 1995. The results showed that the variation of grain protein content and its fractions in somaclones from single cell culture in vitro were very significant and the range was very wide (11531770%). Several types of variation were found in the studies, especially the type with higher protein content than that of cultivar NE 7742 (non-culture parent). Among them, -2069% of lines the grain protein content was significantly higher than that of NE 7742 and combined with high yielding potential. The tendency of variation of the four protein fractions showed that the variation of albumin was not obvious and maintained the same level as NE774 increased in some somaclones and decreased in others. However, the percentages both globulin and glutenin tended to increase. The variation of total amount of structural protein and the ratio between globulin and glutenin tended to increase. The variation of total amount of structural protein and the ratio between globulin and albumm was mainly influenced by globulin under the condition of culture in vitro. The variation of total amount of storage protein and the ratio between gliadin and glutenin was mainly affected by glutenin. The results mentioned above demonstrated that the induction and screening of somaclonal variation could be an effective way in wheat improvement in combining high protein content with high yield.

In vitro culture of immature embryos from Koelreuteria bipinnata var. integrifoliola

For the mass production of Koelreuteria bipinnata var. integrifoliola with selected, hybrid or genetically engineered genotypes, one potentially desirable propagation strategy is based on embryo culture. The immature embryo development in vitro from K. bipinnata var. integrifoliola was studied under different conditions of embryo age, basic culture media and plant growth regulators. The results show that： 1） germination rate of grade 3 embryos in immature seeds with 0.6-0.8 cm diameter was 98.9%. The germination rate of grade 2 embryos in immature seeds with 0.4-0.6 cm diameter was 77,8% and the germination rate of grade 1 embryos in immature seeds with 0.4 cm diameter was 15.6%. 2） The amounts of macroelements in MS medium had no clear effect on the germination rate of immature grade 3 embryos and had a modest effect on plantlet growth, where the best medium was MS or 1/2 MS. The rates were all greater than 90%. 3） The germination rate of grade 3 embryos was greater than 87% when the medium contained a low concentration of NAA or no plant growth regulators at all and decreased markedly when BAP alone or BAP and NAA together were added to the media. We suggest that in vitro culture of immature embryos from K. bipinnata vat. integrifoliola can be enhanced when a small amount of plant growth regulators is added. The addition of BAP has an adverse reaction to the germination and development of immature embryos.

The Key Factors Affecting Tuber Development of Potato in vitro and the Relation with Protein Fractions

According to previous analysis, some properties bounding up with tuber yield were investigated. The results showed that tuber average weight, plastid Mg2 + -ATPase activity, plastid Ca2 + -ATPase activity, mitochondria Mg2 + -ATPase activity, total soluble protein content, tuber average diameter, and Q-enzyme activity were important factors determining the tuber yield. The linear regression equation was:Y = 0.5211 + 0.0595X(1)+0.8389X(2) +0.0882X(3) -0.0073X(4) +0.1449X(5) +0.3510X(6) +0.0031X(7) -0.00003X(8) + 0.3412X(9) + 0.0127X(10) + 0.2904X(ll) + 0.0570X(12) + 0.0159X(13) + 0.3585X(14) + 0.0134X(15) - 0.1012X(16). At the same time, the relation between several important properties and soluble protein fractions were analyzed.

Study on in vitro Cultural Behaviors of Spermatogonium Stem Cells of New Born Calves

Three methods were adopted in culture spermatogoniums of newly born calf in vitro,such as enzymatic digestion and percoll density gradient centrifugation（MethodⅠ）,tubular fragments culture（MethodⅡ）and tissue culture（MethodⅢ）,and cultural behaviors of cells were observed.The results showed that typical spermatogonium colonies appeard at 144 h of culture by enzymatic digestion-percoll density gradient centrifugation method and tubular fragments culture method,2.5%FBS kept the characteristics of spermatogonium stem cell better than others,produced more mass clones,and FBS of more than 2.5%concentration benefited spermatogonium differentiation and the number of colonies was significantly affected by FBS concentration.After 1 week of culture in method Ⅲ,the diameter of lumens and quantity of sertoli’s cells in tubal wall increased obviously,lumen of seminiferous tubules appeared.Sertoli’s cells kept constant and the number of spermatogoniums decreased obviously after 2 weeks of culture.

Normal and Degenerated Rabbit Nucleus Pulposus Cells in in vitro Cultures: A Biological Comparison

This study examined the biological characteristics of normal and degenerated rabbit nucleus pulposus （NP） cells in vitro in order to provide seed cells for intervertebral disc （IVD） tissue engineering. A total of 8 adult New Zealand white rabbits underwent annulus puncture to establish models ofintervertebral disc degeneration （IDD）. Four weeks later, normal and degenerated NP cells were obtained. Cell morphology was observed by light and electron microscopy. Cell viability was measured by MTT assay. Cell cycle and expression of extracellular matrix （ECM）-related genes （aggrecan and type II col- lagen） were determined by using flow cytometry and RT-PCR respectively. The growth curve of normal NP cells showed that the cells at passage 4 tended to slowly grow on the fifth day of culture. The density of normal NP cells at passages 5 to 7 was significantly less than that of the first-passage cells 2 or 3 days after seeding （P〈0.05）. The degenerated NP cells at passage 3 showed slow growth at 4th day. After 5 passages, the degenerated NP cells assumed stagnant growth and the growth seemed to stop at passage 7. The MTT assay revealed that for both normal and degenerated NP cells, the absorbance （.4） value at passages 4-7 was obviously decreased as compared with that at passage 1 （P〈0.05）. Cell cycle analysis showed that the proportion of normal NP cells at G1 phase was 65.4%-3.5%, significantly lower than that of degenerated NP cells at the same cell cycle phase With the value being 77.6%-4.8%. The degen- erated NP cells were predominantly arrested at Gt phase and failed to enter S phase. The expression of type II collagen and aggrecan was significantly decreased with passaging. It was concluded that normal NP cells possessed good viability and proliferative capacity by the third passage, and they could secrete large amounts of ECM within this period. The normal NP cells may serve as seed cells for IVD tissue engineering.

In vitro long-term culture and differentiation of mouse spermatogonia stem cells

The studies from 7 or 8 days old mice were used to prepare the spermatogonial stem cells. The isolation and purification of spermatogonial stem cells were done by means of the Percoll discontinual density gradient concentration. The cells of the 3rd band were collected and cultured in vitro in DMEM supplemented with 2.5% or 10% fetal bovine serum （FBS）. The results showed that with the feeder layer and 2.5% serum, the spermatogonial stem cells could proliferate, differentiate last more than 4 months.