DMSO arrested hybridoma cells for enhanced antibody production / 生物工程学报

Dimethyl sulfoxide (DMSO), a well-known differentiation inducer in several myeloid cells, induces G1 phase arrest in many cell lines. In this study, we investigated the possibility of using DMSO to arrest H18 hybridoma cells to the G1 phase and monitor whether the arrest improves antibody production. We showed that DMSO in concentration ranging between 0.3% and 0.6% efficiently arrested H18 hybridoma cells in G1 phase. In our experiment, > 80% of cells grown for 36h in presence of the 0.6% DMSO were arrested in G1. Furthermore, expression levels of P27 were up-regulated tow fold during the G1 phase. Higher concentration of DMSO at 0.9% leads to cytotoxicity. Herein we show a simple way, a two-stage process for antibody production, which consists of a proliferation phase leading to the desired cell density, followed by an extended production phase during which the cells remain at G1 phase. Our observation that the addition of DMSO results in increase antibody production is of significance in further use of hybridoma cells in high density large scale cell culture.

Bio-panning of antagonistic peptides against HAb18G/CD147 and their function of anti-hepatoma invasion / 中华肿瘤杂志

<p><b>OBJECTIVE</b>To screen out the HAb18G/CD147 binding peptides and find out an antagonist against hepatoma invasion.</p><p><b>METHODS</b>HAb18G/CD147 was purified by affinity chromatographic method and the antigen binding peptides acquired by bio-panning a phage-displayed 12-peptide library. After obtaining the sequence of the selected phage-displayed peptides, all the 9 peptides were synthesized by solid-phase method and identified by mass spectrograph. The peptides' anti-metastatic function was tested by Boyden Chamber assay.</p><p><b>RESULTS</b>The purified HAb18G/CD147, identified by Western blot (molecular weight about 65 kd) could be used to bio-pan the phage-displayed peptide library. After 3 rounds of bio-panning, 9 positive phage clones were selected and sequenced. The synthesized peptides had uneven inhibitory activities and three of them were able to markedly inhibit the hepatoma cell invasion (P < 0.01). The most effective peptide decreased by 90.1% of hepatoma cells migrating through the Boyden Chamber membrane as compared with the control.</p><p><b>CONCLUSION</b>Bio-panning the phage-displayed peptide library can be used successfully to screen out the antigen binding peptides. Hepatoma metastatic potential can be inhibited by peptide antagonist which could be a good foundation of developing peptide therapeutic agent against hepatoma metastasis.</p>

On-line monitoring of oxygen uptake rate and its application in hybridoma culture / 生物工程学报

On-line analysis and control are critical for the optimization of product yields in animal cell culture. The close monitor of viable cell number helps to gain a better insight into the metabolism and to refine culture strategy. In this study, we use the oxygen uptake rate (OUR) to estimate the number of viable cell and the OUR-based feed-back control strategy for nutrients feeding to improve the efficiency of cell culture. A hybridoma cell line (HAb18) was cultured in fed-batch and perfusion model using serum free medium in 5L CelliGen Plus bioreactor (NBS Co., American) and 5L Biostat B bioreactor (Braun Co., Germany). The system and the method for online monitoring OUR in bioreactors, based on the dynamic measurement of dissolved oxygen (DO), were developed. The method of on-line cell concentration estimation was established based on the relationship between the growth of the hybridoma and the uptake rate of oxygen. This method was then used to determine OUR and the concentrations of cell, antibody, glucose, lactate, glutamine and ammonia in the bioreactors at given times. The relationship between OUR and nutrients metabolism was studied and OUR-based feed-back control strategy, which used the state deltaOUR = 0 as the regulation point, was established and used to control the rates of nutrients or medium feeding rate in the perfusion culture. The results showed that there was close relationship between OUR, concentration of live cells, productivity of antibody and consumption of glutamine. The sudden decrease in OUR may be caused by glutamine depletion, and with different delay times, the viable cell concentration and antibody productivity also decreased. The further analysis revealed the linear relationship between OUR and the density of live cells in the exponential growth phase as qOUR = (0.103 +/- 0.028) x 10(-12) mol/cell/h. These findings can be applied to the on-line detection of live cell density. Our study also indicated that by adjusting the perfusion rate with OUR-based feed-back control strategy, it is feasible to continuously increase in viable cell density and antibody concentration in the perfusion culture.

Modification of the antiapoptotic ability of H18 hybridoma cells / 生物工程学报

To construct eukaryotic expression vector containing murine bcl-XL and stably express it in H18 hybridoma cells in order to enhance hybridoma cells antiapoptotic ability. PCR was used to obtain 710bp murine bcl-XL cDNA from pGEM-T-bcl-XL. Then the recombinant expression vector pEF-bcl-XL was constructed by cloning bcl-XL cDNA into eukaryotic expression vector pEF by Pst I and Xho I double digestion. After transfection into H18 hybridoma cells through lipofectamine 2000, the stable expression cell line was screened by 800mg/L G418. The expression of bcl-XL gene was detected by Western blotting. Flow cytometer was used to test the modified hybridoma cells ability to resist apoptosis induced by 0.4mmol/L Sodium Butyrate. The eukaryotic expression vector pEF-bcl-XL was successfully constructed and stably expressed in H18 hybridoma cells. Our data showed that stably transfected H18 cells expressed high levels of Bcl-XL. Under the condition of 0.4mmol/L NaBu, the production of antibody was to be significantly increased by more than 3-fold in stably transfected H18, which resulted from suppressing the NaBu-induced apoptosis and allowing stably transfected H18 cells to grow at higher viability and extend culture longevity by > 3 days. The increased culture longevity by inhibition of NaBu-induced apoptosis by inducible expression of Bcl-XL combined with the enhanced secretion of antibody by NaBu contributed to the enhancement of final antibody concentration in the stably transfected H18 cells culture. The final antibody concentration of stably transfected H18 cells in the presence of NaBu was three-fold higher than that of H18 cells culture in the absence of NaBu. Together, our results showed that butyrate is of practical interest for production of antibody. NaBu-induced apoptosis of hybridoma cells could be inhibited by inducible expression of Bcl-XL. The expression of murine bcl-XL gene in hyridoma cells and the increasing antiapoptosis ability of hybridoma cells are of significance in further use of hybridoma cells in high density large scale cell culture.

Experimental study of corpus cavernosal nerve evoked potential in diabetic rats / 中华男科学杂志

<p><b>OBJECTIVES</b>To observe the cavernosal nerve dysfunction of diabetic Sprague Dawley rats.</p><p><b>METHODS</b>Thirty-five Sprague-Dawley rats were divided randomly into diabetes model group(n = 25) and normal group(n = 10). Diabetes model was established by administration of streptozotocin (63 mg/kg) in single intraperitoneal dosing. Giving single wave stimulus, corpus cavernosal nerve was measured for its latent period of reaction and myopotential.</p><p><b>RESULTS</b>Compared with other groups, diabetic rats had longer reflection latent period(P < 0.01) and higher corpus cavernosum smooth muscle myopotential (P < 0.01).</p><p><b>CONCLUSIONS</b>The results indicate that corpus cavernosal nerve dysfunction may play an important role in the erectile dysfunction of diabetic rats.</p>

The role of vagal innervation in the variability of heart beat / 生理学报

To determine the role of vagi in heart rate variability, conscious rabbits were employed and electrocardiogram was recorded under conditions of bilateral vagi intact, unilateral vagotomy, and bilateral vagotomy. The variability of RR intervals (RRI) was analyzed using power spectrum and approximate entropy (ApEn). The results showed that the values of high frequency power (HF) component, low frequency power (LF) component and ApEn in animals with bilateral vagi intact were the highest, but the LF/HF ratio was the lowest; unilateral vagotomy decreased ApEn, right vagotomy increased LF/HF ratio but left vagotomy did not; the LF/HF ratio increased while ApEn decreased significantly in animals with bilateral vagotomy. It is suggested that the variability of RRI is mainly regulated by the vagi and the role of right vagi is more important than that of the left. When measuring heart rate variability, the results obtained with conventional method are consistent with those with nonlinear method.