A patch-clamp study on human sperm Cl^- channel reassembled into giant liposome

Aim: To record the single-channel currents and characterize the electrophysiological properties of the Cl^- channels inhuman sperm membrane. Methods: The membrane proteins extracted from the human sperm were reassembled intoliposome bilayer, and the liposomes were fused into giant liposomes with a diameter more than 10μm by dehydration-rehydration procedure. The giant liposomes were used to study the Cl^- channel activities by patch-clamp technique.Results: By patch clamping the giant liposome in an asymmetric NMDG (N-methyl-D-glucamine)-Cl (bath 100//pipette 200 mmol/L) solution system, three kinds of single-channel events with unit conductances of (74.1 ± 8.3) pS,(117.0±5.7) pS and (144.7±4.5) pS, respectively, were detected. Their activities were voltage-dependent and allwere blocked by SITS (4-acetamido-4'-isothiocyanato-stilbene-2', 2'-disulfonic acid) in a concentration-dependentmanner. By constructing the open and close dwell time distribution histograms and then fitting them with exponentialfunction, two time constants were obtained in both the open and the close states. The burst activity and conductancesubstate of the channels were observed. Conclusion; There exist three kinds of Cl^- channels with different conduc-tance in human sperm membrane at least. (Asian J Androl 2001 Sep; 3: 185 - 191)

Induction of pancreatic duct cells of neonatal rats into insulin-producing cells with fetal bovine serum: A natural protocol and its use for patch clamp experiments

AIM： To induce the pancreatic duct cells into endocrine cells with a new natural protocol for electrophysiological study. METHODS： The pancreatic duct cells of neonatal rats were isolated, cultured and induced into endocrine ceils with 15% fetal bovine serum for a period of 20 d. During this period, insulin secretion, MTT value, and morphological change of neonatal and adult pancreatic islet cells were comparatively investigated. Pancreatic β-cells were identified by morphological and electrophysiological characteristics, while ATP sensitive potassium channels （KATP）, voltage-dependent potassium channels （Kv）, and voltage-dependent calcium channels （KcA） in β-cells were identified by patch clamp technique. RESULTS： After incubation with fetal bovine serum, the neonatal duct cells budded out, changed from duct-like cells into islet clusters. In the first 4 d, MTT value and insulin secretion increased slowly （MTT value from 0.024 ±0.003 to 3.028±0.003, insulin secretion from 2.6±0.6 to 3.1±0.8 mIU/L）. Then MTT value and insulin secretion increased quickly from d 5 to d 10 （MTT value from 0.028 ±0.003 to 0.052±0.008, insulin secretion from 3.1±0.8 to 18.3±2.6 mIU/L）, then reached high plateau （MTT value 〉0.052±0.008, insulin secretion 〉18.3±2.6 mIU/L）. In contrast, for the isolated adult pancreatic islet cells, both insulin release and MTT value were stable in the first 4 d （MTT value from 0.029±0.01 to 0.031±0.011, insulin secretion from 13.9±3.1 to 14.3±3.3 mIU/L）, but afterwards they reduced gradually （MTT value 〈0.031 ±0.011, insulin secretion 〈8.2±1.5 mIU/L）, and the pancreatic islet cells became dispersed, broken or atrophied correspondingly. The differentiated neonatal cells were identified as pancreatic islet cells by dithizone staining method, and pancreatic β-cells were further identified by both morphological features and electrophysiological characteristics, i.e. the existence of recording currents from KATP, Kv, and KCA. CONCLUSION： Islet cells differentiated from neonatal pancreatic duct cells with the new natural protocol are more advantageous in performing patch clamp study over the isolated adult pancreatic islet cells.

In vivo patch clamp recording technique in the study of neurophysiology

The patch clamp recording technique in vivois a blind patch clamp recording methods to record the current of the spinal or cereral neurons of anaes：hesia （ or awake） animals. This technique can be used to study the synaptic function and plasticity in central nervous system in vivoin order to understand the physiological properties of the ion channels from an integrated point of view. The advantage of this technique have already presented itself in the study of the synaptic transmission and nervous network. Nowadays, in vivo patch whole-cell recording technique in combination with other techniques is becoming a common method in the research fields.

The Reconstructing of Low Signal-noise Ratio Single Ion Channel Signal from Patch-clamp Recordings Sampled in the Colored Background Noise

The single ion channel signal is an ionic current that can be recorded by the patch clamp technique. Hidden Markov model (HMM) algorithm has been used to convert the low signal noise ratio (SNR) noisy recording into an idealized quantal one in the case of white background noise. The traditional HMM algorithm is extended and adapted to the colored background noise. A new algorithm called EHMM (Extended HMM) algorithm is proposed, and mainly validated by simulation. Results show that it’s effective.

Isolation of Protoplast and Ion Channel Recording in Plasma Membrane of Suspension Cells of Populus euphratica

The authors used suspension cells of Populus euphratica to isolate protoplast in the present study. Protoplasts were successfully obtained after 4 hours incubation in enzyme solution containing 1 0% cellulase “onozuka” R\|10, 0\^01% pectolyase Y\|23,0\^15% macerozyme R\|10 and 0\^1% hemicellulase at 25℃. Outward and inward single channels in plasma membrane were observed using cell\|attached recording of patch\|clamp technique. In this study, single channel records showed that more than one species of channel were obtained. These attempts in protoplast isolation and ion channel recording offers the opportunity to characterize cellular mechanisms of salt tolerance in tree species.

Effects of IQ_23 on the Repolarization and Both Components of the Delayed Rectifier Potassium Current in Guinea Pig Ventricular Myocytcs

Using patch clamp whole cell recording techiques, we examined the effects ofIQ_23, a benzyl-isoquinoline derivative with antiarrhythmic activities, on the action potential (AP) andpotassium currents in single guinea pig ventricular myocytes. The results showed that IQ_23 at 10, 30and 100 μmol ·L_-1 slowed the repolarization in AP dose-dependently. The APD_90 were prolonged by15%, 28% and 31% respectively. This effect did not depend on the extracellular Ca^2+. In voltageclamp mode, IQ_23 effectively blocked both the components of the delayed rectifier potassium current(I_k), i.e., I_ks and I_kr. At concentrations of 30 and 100 μmol· L^-1, IQ_23 suppressed I_ks by 21% and 26%and suppressed I_kr by 67% and 86% respectively. But even at 100 μmol·L^-1, IQ_23 had little effect onthe inward rectifier potassium current (I_k1). It is concluded: 1. IQ_23 can dose-dependently prolongAPD in the ventriculas myocytes of guinea pig, the effect does not depend on the extracellular Ca^2+; 2.IQ_23 blocks both I_ks and Ikr in the ventricular myocytes without obvious specificities between them.

Effects of Rhynchophylline on L type Calcium Channels in Isolated Rat Cortical Neurons During Acute Hypoxia

Aim: To study the effects of rhynchophylline (Rhy) on the L type calcium channels in freshly dissociated cortical neurons of Wistar rats during acute hypoxia. Methods: Cell attached configuration of patch clamp technique. L type calcium channel was activated by stepping from 40 mV to 0 mV. Results: The results showed that the L type calcium channels of cortical neurons were activated by acute hypoxia. The mean open time of the channel was increased, the mean close time decreased and the open state probability raised during acute hypoxia. Rhy (15 and 30μmol·L -1 ) in concentration dependent manner blocked activity of the channels. The drug shortened the mean open time of the channels from 8 87 ms to 3 03 ms and 2 23 ms ( P 【0 001), prolonged the mean close time from 9 23 ms to 38 84 ms and 54 43 ms ( P 【0 001), and decreased the open state probability from 0 142 to 0 031 and 0 025 ( P 【0 001) under the hypoxia condition, respectively. The effects of Rhy were similar to but weaker than those of verapamil (15 μmol·L -1 ). Conclusion: The study confirmed that Rhy has the blockade effects on L type calcium channels in cortical neurons of rats during hypoxia, by which it protects the brain from hypoxic injury.

The Effect of Benzyltetrahydropalmatine (BTHP) on Action Potentials and the Two Components of Delayed Rectifying Potassium Currents in Guinea Pig Ventricular Myocytes

The effects of BTHP on Ca 2+ independent action potential and the two components of delayed rectifier potassium currents were studied in guinea pig single ventricular myocytes by using whole cell patch clamp technique. BTHP 30 μmol·L -1 significantly prolonged APD 90 from 143±16 ms to 184±21 ms ( P 【0.01, n=5) without affecting either the RP or APA, and the APD prolonging effects of BTHP were independent of extracellular Ca 2+ . BTHP inhibited both I kr (IC 50 =7 9 μmol·L -1 ) and I ks (IC 50 =22 4 μmol·L -1 ) in a concentration dependent fashion. The results demon strated that BTHP had no obvious selectivity for I kr and I ks .

Effects of IQ23 on the Repolarization and Both Components of the Delayed Rectifier Potassium Current in Guinea Pig Ventricular Myocytcs

Using patch clamp whole cell recording techiques, we examined the effects ofIQ23, a benzyl-isoquinoline derivative with antiarrhythmic activities, on the action potential （AP） andpotassium currents in single guinea pig ventricular myocytes. The results showed that IQ23 at 10, 30and 100 μmol ·L-1 slowed the repolarization in AP dose-dependently. The APD90 were prolonged by15%, 28% and 31% respectively. This effect did not depend on the extracellular Ca2+. In voltageclamp mode, IQ23 effectively blocked both the components of the delayed rectifier potassium current（Ik）, i.e., Iks and Ikr. At concentrations of 30 and 100 μmol· L-1, IQ23 suppressed Iks by 21% and 26%and suppressed Ikr by 67% and 86% respectively. But even at 100 μmol·L-1, IQ23 had little effect onthe inward rectifier potassium current （Ik1）. It is concluded: 1. IQ23 can dose-dependently prolongAPD in the ventriculas myocytes of guinea pig, the effect does not depend on the extracellular Ca2+; 2.IQ23 blocks both Iks and Ikr in the ventricular myocytes without obvious specificities between them.

K^+channels inhibited by hydrogen peroxide mediate abscisic acid signaling in Vicia guard cells

A number of studies show that environmental stress conditions increase abscisic acid (ABA) and hydrogen peroxide (H2O2) levels in plant cells. Despite this central role of ABA in altering stomatal aperture by regulating guard cell ion transport, little is known concerning the relationship between ABA and H2O2 in signal transduction leading to stomatal movement. Epidermal strip bioassay illustrated that ABA- inhibited stomatal opening and ABA-induced stomatal closure were abolished partly by externally added catalase (CAT) or diphenylene iodonium (DPl), which are a H2O2 scavenger and a NADPH oxidase inhibitor respectively. In contrast, internally added CAT or DPI nearly completely or partly reversed ABA-induced closure in half-stoma. Consistent with these results, whole-cell patch-clamp analysis showed that intracellular application of CAT or DPI partly abolished ABA-inhibited inward K+ current across the plasma membrane of guard cells. H2O2 mimicked ABA to inhibit inward K+ current, an effect which was reversed by the addition of ascorbic acid (Vc) in patch clamping micropipettes. These results suggested that H2O2 mediated ABA-induced stomatal movement by targeting inward K+ channels at plasma membrane.

Cranial irradiation impairs intrinsic excitability and synaptic plasticity of hippocampal CA1 pyramidal neurons with implications for cognitive function

Radiation therapy is a standard treatment for head and neck tumors.However,patients often exhibit cognitive impairments following radiation therapy.Previous studies have revealed that hippocampal dysfunction,specifically abnormal hippocampal neurogenesis or neuroinflammation,plays a key role in radiation-induced cognitive impairment.However,the long-term effects of radiation with respect to the electrophysiological adaptation of hippocampal neurons remain poorly characterized.We found that mice exhibited cognitive impairment 3 months after undergoing 10 minutes of cranial irradiation at a dose rate of 3 Gy/min.Furthermore,we observed a remarkable reduction in spike firing and excitatory synaptic input,as well as greatly enhanced inhibitory inputs,in hippocampal CA1 pyramidal neurons.Corresponding to the electrophysiological adaptation,we found reduced expression of synaptic plasticity marker VGLUT1 and increased expression of VGAT.Furthermore,in irradiated mice,long-term potentiation in the hippocampus was weakened and GluR1 expression was inhibited.These findings suggest that radiation can impair intrinsic excitability and synaptic plasticity in hippocampal CA1 pyramidal neurons.

Neuroprotective effect of interleukin-6 regulation of voltage-gated Na^+ channels of cortical neurons is time-and dose-dependent

Interleukin-6 has been shown to be involved in nerve injury and nerve regeneration, but the effects of long-term administration of high concentrations of interleukin-6 on neurons in the central nervous system is poorly understood. This study investigated the effects of 24 hour expo-sure of interleukin-6 on cortical neurons at various concentrations （0.1, 1, 5 and 10 ng/mL） and the effects of 10 ng/mL interleukin-6 exposure to cortical neurons for various durations （2, 4, 8, 24 and 48 hours） by studying voltage-gated Na＋ channels using a patch-clamp technique. Volt-age-clamp recording results demonstrated that interleukin-6 suppressed Na＋ currents through its receptor in a time- and dose-dependent manner, but did not alter voltage-dependent activation and inactivation. Current-clamp recording results were consistent with voltage-clamp recording results. Interleukin-6 reduced the action potential amplitude of cortical neurons, but did not change the action potential threshold. The regulation of voltage-gated Na＋channels in rat corti-cal neurons by interleukin-6 is time- and dose-dependent.

Whole-cell recordings of calcium and potassium currents in acutely isolated smooth muscle cells

AIM： To record calcium and potassium currents in acutely isolated smooth muscle cells of mesenteric arterial branches in rats. METHODS： Smooth muscle cells were freshly isolated by collagenase digest and mechanical trituration with polished pipettes. Patch clamp technique in whole-cell mode was employed to record calcium and potassium currents. RESULTS： The procedure dissociated smooth muscle cells without impairing the electrophysiological characteristics of the cells. The voltage-gated Ca^2＋ and potassium currents were successfully recorded using whole-cell patch clamp configuration. CONCLUSION： The method dissociates smooth muscle cells from rat mesenteric arterial branches. Voltage-gated channel currents can be recorded in this preparation.

Extract from Buthus martensii Karsch is associated with potassium channels on glioma cells

Catilan extracted from Leiurus quinquestriatus is a specific ion channel blocker.It can specifically bind chloride channels of glioma cells and kill these tumor cells.The questions remain as to whether antigliomatin,the extract from scorpion venom of Buthus martensii Karsch in China,can inhibit glioma growth,and whether this inhibition is correlated with ion channels of tumor cells.The present study treated rat C6 glioma cells with 0.8,1.2,and 1.6 μg/mL antigliomatin for 20 hours.Whole-cell patch clamp technique showed that antigliomatin delayed rectifier potassium channels of C6 glioma cells.Antigliomatin inhibited tumor growth,which could potentially involve potassium channels of tumor cells.

Effects of Atractylodes Macrocephala on the Cytomembrane Ca^(2+)-activated K^+ Currents in Cells of Human Pregnant Myometrial Smooth Muscles

The study examined the inhibitory effect of Atractylodes macrocephala （AM） on the uterine contraction during premature delivery and explored its electrophysiological mechanism by studying the effects of AM on the Ca^2＋-activated K^＋ currents of pregnant human myometrial smooth muscle cells with or without the treatment with intedeukin-6. Single cells were acutely isolated from pregnant human myometrial smooth muscles. Whole-cell Ca^2＋-activated K^＋ currents were recorded by using an Axopatchl-D amplifier. The cells were divided into three groups： group A in which AM was added into perfusate, group B, in which interleukin-6 was added into perfusate） and group C in which AM was added into perfusate after addition of interleukin-6. IL-6 10 ng/mL inhibited BKca by 36.9%±13.7% as compared with control （P〈0.01）. AM at 2 mg/mL raised BKca by 36.7%±22.6% or 45.2%±13.7% with or without the treatment of IL-6, respectively （P〈0.01）. It is concluded that AM was able to enhance the BKca of pregnant human myometrial smooth muscle cells treated or untreated with interleukin-6 and its effect on the BKca IL-treated cells was stronger that its effect on BKca of untreated cells. Our results suggested that AM can help to maintain the membrane potentials and the resting status of pregnant human myometrial smooth muscle cells.

Expression of gamma-aminobutyric acid type A receptor α_2 subunit in the dorsal root ganglion of rats with sciatic nerve injury

The γ-aminobutyric acid neurotransmitter in the spinal cord dorsal horn plays an important role in pain modulation through primary afferent-mediated presynaptic inhibition. The weakening of γ-aminobutyric acid-mediated presynaptic inhibition may be an important cause of neuropathic pain. γ-aminobutyric acid-mediated presynaptic inhibition is related to the current strength of γ-aminobutyric acid A receptor activation. In view of this, the whole-cell patch-clamp technique was used here to record the change in muscimol activated current of dorsal root ganglion neurons in a chronic constriction injury model. Results found that damage in rat dorsal root ganglion neurons following application of muscimol caused concentration-dependent activation of current, and compared with the sham group, its current strength and γ-aminobutyric acid A receptor protein expression decreased. Immunofluorescence revealed that γ-aminobutyric acid type A receptor α2 subunit protein expression decreased and was most obvious at 12 and 15 days after modeling. Our experimental findings confirmed that the y-aminobutyric acid type A receptor α2 subunit in the chronic constriction injury model rat dorsal root ganglion was downregulated, which may be one of the reasons for the reduction of injury in dorsal root ganglion neurons following muscimol-activated currents.

Gamma-knife for treating pituitary adenomas in 501 cases: Retrospective case analysis

BACKGROUND： Gamma-knife is characterized by low risk and low death rate, without trauma, bleeding or infection. MRI has replaced CT as a method of location, and provides a good choice for treating pituitary tumors.OBJECTIVE： To analyze the effectiveness and complications of 501 cases with pituitary adenoma by using gamma-knife retrospectively.DESIGN： Case-analysis.SETTING： Gamma-knife Center, the 363 Hospital of Chinese PLA.PARTICIPANTS： A total of 501 cases were selected from Gamma-knife Center, the 363 Hospital of Chinese PLA from January 1997 to December 2002. All patients were certainly diagnosed with CT and MRI scanning. There were 186 males and 315 females. Their ages ranged from 15 to 84 years with the mean age of 39 years. All patients provided confirmed consent.METHODS： All 501 cases with pituitary adenomas were treated by gamma-knife or combined with operations： The average iso-dose curve was 50% （30% - 65%）, peri-dose was 14 Gy and the average target number was 5.17. ① At 35 months after treatment, patients received follow-up including clinical symptoms,imaging symptoms and endocrine symptoms. ② Patients who received second gamma-knife treatment were analyzed and their complications were observed after operation.MAIN OUTCOME MEASURES： ① Follow-up results of clinical symptoms, image and endocrine indexes; ② second gamma-knife treatment; ③ postoperative complications.RESULTS： Among 501 accepted patients, 275 cases were involved in the follow up of clinical symptoms,154 in image symptoms and 98 in endocrine symptoms. ① Follow-up results of clinical symptoms, image and endocrine indexes： Follow up of clinical symptoms demonstrated that clinical symptoms of 169 cases were relieved, of 68 disappeared, and of 38 deteriorated. Follow-up of image symptoms indicated that pituitary tumor of 25 cases disappeared, of 84 shortened, of 42 not changed, and of 3 enlarged. Follow-up of endocrine symptoms suggested that endocrine of 50 cases was abnormal, of 29 recovery to normal value, and of 19 not changed obviously. ② Second gamma-knife treatment： At 6- 24 months after gamma-knife treatment, 15 patients with pituitary tumor received second gamma-knife treatment; especially, one patient received for the three times. The conditions in details were recorded as follows： The peri-dose for the first treatment ranged from 8 to 12 Gy; in addition, that for the second one ranged from 6 to 12 Gy. ③ Postoperative complications： The complications were as follow： hypopituitarism occurred in 2 patients （0.7%）; tumor apoplexy in 2 patients （0.7%）;weakened eyesight in 5 patients （1.8%）; 3 of which recovered with dehydration and hormone treatment; 2 patients received tumor resection and optical nerve decompression operation with dehydration and hormone treatment ineffectively.CONCLUSION： Gamma-knife is effective and safe for pituitary adenoma.

Tyrosine hydroxylase-positive cells and dopaminergic neuronal function in human embryonic stem cells: An electrophysiological validation

BACKGROUND： Induced differentiation strategies and cytochemical properties of human embryonic stem ceils （hESCs） have been investigated. However, the electrophysiological functions of tyrosine hydroxylase （TH）-positive cells dedved from hESCs remain unclear. OBJECTIVE： To investigate the differentiation efficiency of TH-positive cells from hESCs in vitro using modified four-step culture methods, including embryoid body formation, and to examine the functional characteristics of the differentiated TH-positive cells using electrophysiological techniques. DESIGN, TIME AND SETTING： Neuroelectrophysiology was performed at the Reproductive Medicine Center and Stem Cell Research Center, Peking University Third Hospital, and the Neuroscience Research Institute and Department of Neurobiology, Peking University, from September 2004 to August 2008. MATERIALS： The hESC line, PKU-1.1, a monoclonal cell line derived from a pre-implantation human blastocyst in the Reproductive Medical Center of Peking University Third Hospital. The patch clamp recording system was provided by the Neuroscience Research Institute and Department of Neurobiology, Peking University. METHODS： The hESC line was induced to differentiate into TH-positive cells in vitro using a modified four-step culture method, including the formation of embryoid body, as well as the presence of sonic hedgehog and fibroblast growth factor 8. The cell karyotype was assessed by G-banding karyotype analysis techniques and specific markers were detected immunocytochemically. Whole-cell configuration was obtained after obtaining a tight seal of over 1 GΩ. Ionic currents were detected by holding the cells at -70 mV and stepping to test voltages between -80 and 40 mV in 10-mV increments in voltage-clamp configuration. MAIN OUTCOME MEASURES： We measured the cell karyotype, specific cell markers, and the electrophysiological properties of the voltage-gated ion channels on the cell membrane of TH-positive dopaminergic cells differentiated from our hESCs line in vitro. RESULTS： The differentiated cells had a consistent appearance, and the majority of cells （〉 90%） expressed TH and β-tubulion, as well as the neural progenitor marker, nestino Cell karyotype analysis demonstrated that all of the hESCs had a stable and normal karyotype （46, XX） after differentiation. In addition, patch clamp recording showed that the 10 recorded TH-positive cells exhibited a fast inward current when the test voltage depolarized to -30 mV, and a delayed outward current when the test voltage depolarized to -10 mV. The peak of inward current was obtained at voltage between 10 mV and 0 mV, while the peak of outward current was obtained at 40 mV. The average peak of inward current density was （ -50.05 ± 15.50） pA/pF, and the average peak of outward current density was （41.98 ± 13.55） pA/pE CONCLUSION： More than 90% of the differentiated hESC-derived cells induced by the modified four-step culture method exhibit dopaminergic neuronal properties, including general electrophysiological functional properties, such as functional potassium and sodium channels.

Effect of Chengzai Pill on the L-Type Ca^(2+) Channel Currents of Osteoblasts Pretreated with Methylprednisolone

Objective: To observe the effect of the serum containing Chengzai Pill on the L-type voltage-sensitive calcium channels current (L-VSCCsC) of osteoblastic MC3T3-E1 cells pretreated with methylprednisolone (mPSL). Methods: A control group, a model group, a low dose group and a high dose group were set up. The whole cell patch clamp technique was used to record L-VSCCsC of 10 osteoblastic MC3T3-E1 cells in each group and their peak currents were determined. Results: The peak current of the control group was 0.2284±0.0209 nA; the peak current of the model group was 0.1839±0.0179 nA; decreased by 19.5% as compared with the control group (P<0.01); the peak current of the low and high dose groups was 0.2526± 0.0093 nA and 0.2671±0.0120 nA respectively, increased by 37.4% and 45.2% as compared with the model group (P<0.01); the difference between the low and high dose groups was P<0.05. Conclusion: 1. mPSL inhibits L-VSCCsC of osteoblasts; and 2. The serum containing Chengzai Pill increases L-VSCCsC of osteoblasts pretreated with mPSL.

MicroRNA is a potential target for therapies to improve the physiological function of skeletal muscle after trauma

MicroRNAs can regulate the function of ion channels in many organs.Based on our previous study we propose that miR-142a-39,which is highly expressed in denervated skeletal muscle,might affect cell excitability through similar mechanisms.In this study,we overexpressed or knocked down miR-142a-3p in C2C12 cells using a lentivirus method.After 7 days of differentiation culture,whole-cell currents were recorded.The results showed that overexpression of miR-142a-3p reduced the cell membrane capacitance,increased potassium current density and decreased calcium current density.Knockdown of miR-142a-3p reduced sodium ion channel current density.The results showed that change in miR-142a-3p expression affected the ion channel currents in C2C12 cells,suggesting its possible roles in muscle cell electrophysiology.This study was approved by the Animal Ethics Committee of Peking University in July 2020(approval No.LA2017128).