0.05).IHC loss was found under hypoxia for 2 h to 48 h in a time dependent manner,the cell density in unit area had remarkable difference compared with control(P

Objective To investigate the correlation of cochlear injury and expressions of Bcl-2 and Bax in the cochlea of diabetic rats.Methods Thirty five Wistar rats were randomly divided into diabetic group(n=25) and control group(n=10).Diabetic deafness was induced in rats by intraperitoneal injection of streptozotocin,while the rats in normal group were injected with normal saline.Immunohistochemical staining was used to detect the protein levels of Bcl-2 and Bax in the cochlea after 3 months.Results No significantly morphological change in cochlea of diabetic rats was found.The Bcl-2 levels in stria vascularis in diabetic group and control group were 135.32?21.69 and 45.32?9.08,respectively,there was significant difference(P

Objective To investigate the protective effects of magnesium sulfate on glutamate-induced neurotoxicity in cultured cochlear.Methods The cortis of newborn rats were cultivated in vitro. The cultured cochlear neurons were divided into glutamate group treated with 0.1 mmol?L-1 glutamate,magnesium sulfate group treated with different concentrations of magnesium sulfate(0.5,1.0,2.0 and 4.0 mmol?L-1) and combined group treated with glutamate and magnesium sulfate for 24 h. Immunofluorescence staining of Neurofilament 200kDa antibody was used to label spiral ganglion neuron(SGN) and the terminate of dendrites. The morphological changes of SGN were examined by fluorescent microscope and the amount of SGN was determined. In addition,the SGN was loaded with 10 ?mol?L-1 Fluo-3 /AM,and the intracellular Ca2+was measured by laser confocal scanning microscope (LCSM). Results Many SGN were smaller and swelling and vacuoles at the terminate of dendrites following glutamate treatment for 24 h. In combined group treated with glutamate and 0.5,1.0 and 2.0 mmol?L-1 of magnesium sulfate,the amounts of SGN increased compared with glutamate group(P

Linoleic acid (C18∶2n-6) and ?-linolenic acid (C18∶3n-3) are found widely in fungi, plants and some lower animals. However, they can not be synthesized in mammals due to lack of △12 and ?-3 fatty acid desaturases. To enable endogenous production of essential fatty acids in mammalian cells, here the stable expression of a Caenorhabditis elegans gene FAT-2 encoding △12 fatty acid desaturase in CHO cells was reported. First, the FAT-2 coding sequence was cloned by RT-PCR. To facilitate high level synthesis of heterogeneous protein, the codon usage of the fatty acid desaturase genes was optimized according to the codon preference of mouse by site-directed mutagenesis, 2 synonymous mutations were introduced into FAT-2 gene by overlapping PCR. The codon-modified gene was finally fused to pBudCE4.1 vector (Invitrogen) under the control of CMV promoter. The expression vector pBudCE-FAT2 was linearized with NheⅠ, and then transfected CHO cells, the cells were under Zeocin selection for nine days and then propagated, then the transfected cells were harvested. The genome and total RNA were isolated for PCR and Norhern blot ananlysis. The results revealed that FAT-2 gene has been integrated into the genome of CHO cells and expressed properly. Fatty acids of total cellular lipids were analyzed by gas chromatography. The results indicate that the expression and function of △-12 fatty acid desaturase resulted in accumulation of linoleic acid. The levels of linoleic acid in transgenic cells were 2.4-fold higher than those in wild-type cells. The moderate linoleic acid in CHO cells was derived from cell culture media uptaken by cell membrane. The results demonstrate that a heterogenous desaturase gene can function well in mammalian cells and prove that transgenic approach is an efficient strategy for changing fatty acid composition of mammals.

BACKGROUND: Studies are very few regarding the specific reaction of bone marrow mesenchymal stem cells (BMSCs) to activated microglia. Moreover, it remains unclear how MSCs maintain dopaminergic neuronal survival under specific microenvironment.OBJECTIVE: To explore the effect of BMSCs stimulated by activated microglia on dopaminergic neuron survival.METHODS: BMSCs were isolated from Wistar rats by attachment method, and in vitro cultured; microglia was activated, and dopaminergic neurons were cultured by enzyme digestion method. The experiment included 5 groups: BMSCs, microglia, lipopolysaccharide (LPS)+microglia; BMSCs+LPS+microglia groups, in which the dopaminergic neurons were cultured with corresponding culture medium; the dopaminergic neurons alone group was cultured with 10% fetal bovine serum+ DMEM/F12. The effect of different microenvironment on dopaminergic neuron survival and gliocyte-derived neurotrophic factor released from BMSCs were detected by immunofluorescence technique.RESULTS AND CONCLUSION: The release of gliocyte-derived neurotrophic factor in groups involving BMSCs was greater than corresponding control group. Tyrosine hydroxylase immunofluorescence showed that neuronal survival of dopaminergic neurons alone group was 15%, microglia group was 10%, LPS+microglia was 5%, but BMSCs+LPS+microglia group was 28%, significantly greater than the other groups (P < 0.05). In addition, survival of in vitro cultured dopaminergic neurons was decreased with increasing culture duration, but the survival of dopaminergic neurons in group involving BMSCs was significantly greater than corresponding control group. This indicates that microglia activation stimulated BMSCs to upregulate gliocyte-derived neurotrophic factor to prevent dopaminergic neurons from toxic injury, and inhibit delayed death of dopaminergic neurons.

Vaccination has been proved to be the most effective strategy to prevent the Corona Virus Disease 2019 (COVID-19). The mRNA vaccine based on nano drug delivery system (NDDS) - lipid nanoparticles (LNP) has been widely used because of its high effectiveness and safety. Although there have been reports of severe allergic reactions caused by mRNA-LNP vaccines, the mechanism and components of anaphylaxis have not been completely clarified yet. This review focuses on two mRNA-LNP vaccines, BNT162b2 and mRNA-1273. After summarizing the structural characteristics, potential allergens, possible allergic reaction mechanism, and pharmacokinetics of mRNA and LNP in vivo, this article then reviews the evaluation methods for patients with allergic history, as well as the regulations of different countries and regions on people who should not be vaccinated, in order to promote more safe injection of vaccines. LNP has become a recognized highly customizable nucleic acid delivery vector, which not only shows its value in mRNA vaccines, but also has great potential in treating rare diseases, cancers and other broad fields in the future. At the moment when mRNA-LNP vaccines open a new era of nano medicine, it is expected to provide some inspiration for safety research in the process of research, development and evaluation of more nano delivery drugs, and promote more nano drugs successfully to market.

Objective:Mutations in the CX(connexin)26 gene (GJB2), encoding the gap\|junction protein. Connexin 26, have been shown to be the major cause of non\|syndromic recessive deafness. Among these mutations, the CX 26 cDNA (35delG) mutation accounts for the majority of this kind of deafness are European and America, but it does not present in China. Therefore, we intend to investigate the mutation hot spot of connexin 26 in China deafness populations. Mathods:The mutation of 233delC was detected by the technique of PCR\|RFLP and analyzed by directly sequencing protocols in 219 patients with kinds of deafness and 50 individuals with normal hearing. Results: The frequency of the 233delC mutation was 47/219(21.5%) in the Chinese population. No mutation was found in group with normal hearing and dominant nonsyndromic hearing loss. The mutation frequency for 233delC was 4 in 15 patients (26.7%) with genetic prelingual deafness and 10 in 50 patients(20.0%) with drug\|deafness. Conclusion: The 233delC mutaion had a high frequency in Chinese population with hearing loss, especially congenital deafness. Our data indicated that specific combinations of GJB2 mutation exist in different populations.

OBJECTIVE: To construct an adenoviral vector that codes for both human NT3 and EGFP, to confirm the transduction efficiency in rat cochlear cultures and to assess the protection of NT3 on SGNs survival. METHOD: PAdeasy-1 and pAdTrack CMV were used to constructed Ad/NT3 adenovirus and then to transfer postnatal day 3 rat cochlear cultures. The transduction efficiency was determined by microscope observation. The amounts of SGNs were counted to evaluated protection of Ad/NT3 on SGNs survival. RESULT: EGFP positive cells were observed in all cochlear turns. There was approximately 49% in outer sulcus cells and 27% in the interdental cells; less than 2% of the hair cells and SGN. The amounts of SGN of treated Ad/NT3 adenovirus are more than cochlea SGN only Ad/EGFP adenovirus after cultured for 15 days. CONCLUSION Ad/NT3 adenovirus could transduce EGFP and NT3 in large number of supporting cells, but few hair cells or SGNs. The putative release of NT3 from these supporting cells could enhance cell survival and promote neurite outgrowth from SGNs.
Adenoviridae ; genetics ; Animals ; Basilar Membrane ; cytology ; Cell Survival ; genetics ; Cells, Cultured ; Cochlea ; cytology ; Genetic Vectors ; Hair Cells, Auditory ; cytology ; Humans ; Neurotrophin 3 ; genetics ; Rats ; Rats, Inbred F344 ; Transfection

Objective To investigate the effects of high-affinity tyrosine kinase receptors TrkB,TrkC and the low-affinity neurotrophin receptor p75 in spiral ganglion cell(SGC)of cisplatin-induced ototoxicity.Methods The 50 adult Wistar rats were divided randomly into 5 groups received intraperitoneal injection of cisplatin with vary dose.Control group was received equivalent volumes of saline.The group received l day intraperitoneal injection was cisplatin treated at a dose of 5 mg/kg and killed at next day.The group received 3 days was cisplatin treated for 3 days at sanle dose daily and then killed at next day.The group A received 5 days was cisplatin treated for 5 days and killed at next day.The group B received 5 days was cisplatin treated for 5 days and then were sacrificed after 7 davs.The change of mRNA level of neurotrophin receptors in cochlear tissue were examined bv RT-PCR.The expressing pattern of TrkB,TrkC,P75 in damaged cochlea were study by immunochemistry using antibodies against TrkB,TrkC,P75 protein.Results The research data showed the expression of Trk B,Trk C,D75 exhibited in SGC wag dynamic along with the administration lasting.The mRNA and protein level of Trk B(x±s)at day 1 and 3 after cisplatin treatment were 0.76±0.06,88.78±4.28,0.82±0.09 and 91.64±4.06,with significant difference among those and other groups(P＜0.05).The mRNA and protein level of TrkC at day 1 after cisplatin treatment were 0.80±0.05 and 89.55±2.76.with significant difference among that and other groups(P＜0.05).The mRNA and protein level of p75 at the control group and cisplatin treated groups were 0.64±0.04,55.16±3.10.0.77±0.04,78.46±3.86,1.01±0.09,105.02±6.61,1.18±0.09,111.10±6.08.0.51±0.04 and 42.74±5.20.with significant difference among the control group and cisplatin treated groups(P＜0.05).Conclusions The expression of Trk B increased to peak at day 1-3 after cisplatin treatment and decreased at day 5 early and following weeks.The expression of Trk C went up to peak at day l after cisplatin treatment and went down during subsequently time.P75 kept a trend of continuance increased during the drug treatment and decrease at drug stopped.The expression of Trk B.Trk C and P75 may be involved in cochlear insuh with cisplatin-induced.Trk B and Trk C may play an important role in the reparative process of cochlear,especially at early stage of the damage.P75 could promote SGC apoptosis in cisplatin-induced neuretoxicity.

OBJECTIVE: To establish a practical model for Wistar rat cochlea organ culturing in vitro, and to observe the growing status in hypoxia of the spiral ganglion cell and nerve fiber. METHOD: We used an in vitro hypoxia model and dissociated cultures of the basal membrane from the cochlea of 3-day-old Wistar rats. And put them in incubator (37 degrees C, 90% N2, 5% CO2, 5% O2) to hypoxia culture for different times. The culture were Immunofluorescence dyed and count the number of the spiral ganglion cell and the cell density in unit area (24 mm x 36 mm), and observe the morph of nerve fiber under the confocal microscope, the results were compared with controls. RESULT: Hypoxia early (6 h) nerve fiber appear edema, spiral ganglion cell didn't change compared with controls; nerve fiber appear break and disintegration and the spiral ganglion cell decrease in 12 hours culturing, and the cell density in unit area had remarkable difference compared with control (P < 0.01). Hypoxia leads to the cell density decrease in a time-dependent manner, the longer of cultures times in hypoxia, the heavier of damage in spiral ganglion cell and nerve fiber. Twelve hours culturing, and the cell density in unit area had remarkable difference compared with control (P < 0.01). Hypoxia leads to the cell density decrease in a time-dependent manner, the longer of cultures times in hypoxia, the heavier of damage in spiral ganglion cell and nerve fiber. CONCLUSION The study findings suggest that hypoxia makes the spiral ganglion cell and nerve fiber damage of culturing in vitro, and nerve fiber more susceptible than spiral ganglion cell for hypoxia.
Animals ; Cell Count ; Cell Survival ; Female ; Hair Cells, Auditory, Inner ; cytology ; pathology ; Hypoxia ; pathology ; In Vitro Techniques ; Male ; Nerve Fibers ; pathology ; Rats ; Rats, Wistar ; Spiral Ganglion ; cytology ; pathology