Space Life Science of China*

MA Hong CHEN Yu REN Hao LI Xiaoqiong YANG Chunhua LI Bo,2 HAN Chu,2 ZHANG Ying LI Yujuan LONG Mian ZHUANG Fengyuan,4 DENG Yulin

Space Life Science of China*

MA Hong1CHEN Yu1REN Hao1LI Xiaoqiong1YANG Chunhua1LI Bo1,2HAN Chu1,2ZHANG Ying1LI Yujuan1LONG Mian3ZHUANG Fengyuan1,4DENG Yulin1

1 (1000812 (100081) 3(100190) 4(100191)

In the past two years, China’s space life science has made great progress. Space biomedical and life science programs have carried out ground-based research for the first batch of projects, and are preparing to carry out space-based experiments along with the construction of China’s space station. And space life science payload of the space station completed the development of positive samples. Thus, with the development of lunar exploration and Mars exploration projects, astrobiology research has also made a lot of basic achievements. On the basis of summarizing the development of space life science in China, this paper mainly introduces the important progress of payload technology and life science research.

Space life sciences, Space payload, Simulated microgravity, Ionizing radiation

Space life science is a discipline that studies the life phenomena and their laws under the action of special environmental factors of space (such as vacuum, high or low temperature, microgravity, and cosmic radiation,.). Broadly speaking, it includes space biology, space physiology, space medicine, and space bioengineering. It belongs to the edge discipline of space science and life science, and is also a newly formed branch of space science. If human beings want to live in the space for a long time and develop the space, we need to study and solve a series of space life problems induced by cosmic environmental factors on the life process. Here, we review and summarize the researches on space life sciences which were con-tributed by Chinese scientists in the last two years.

1 Payloads Technology in Space Life Science

1.1 Automatic Device for Cell Culture in Space Based on Microfluidic Chip

Mammalian cell culture is one of the commonest methods in life science research. In space, cell culture was commonly carried out in T-flask, coverslips, Teflon culture bag or other traditional tools. These tools are lowly integrated and disable to precisely control the environment around the cells. In recent decades, microfluidic chip was applied to research in space biology. Microfluidic chip offers promising solutions for mammalian cell culture and precise con-trol of the extracellular microenvironment in vitro.

Therefore, a device for mammalian cell culture in microfluidic chip was developed for space biology research[1]. The device consists of a cell culture unit based on microfluidic chip, microscope imaging system, injection pump, reagents control unit, waste storage unit, central control unit. Mammalian cells were cultured in a single-chamber microfluidic chip placed in a cell culture unit that can control the temperature of chip, commonly at 37℃. Injection pump was connected with the microfluidic chip to perfuse the fresh cell culture medium. Using valve and pump group in reagents control unit, the reagents can be injected into microfluidic chip, such as phosphate buffer saline (PBS) buffer solution, trypsine solution, dye solution,. Thus, cells in the chip can be trypsinized, dyed or lysed. Then the cells or cell lysates can be collected for further analysis. During the experiment, cells can be observed constantly by microscope imaging system, capable of recording the cell morphology change during the whole experiment.

Some experiments were carried out to test the performance of the device. Firstly, the thermal control ability was analyzed. The results show that the temperature of cell culture unit was 37±0.5℃. Then the analysis of resolution of the microscope imaging system was carried out. The results showed that the resolution was higher than 2 μm. Finally, U87-MG cells were cultured then dyed by eosin and imaged by a microscope imaging system. In summary, this device, integrated cell culture, microscope imaging, and cell sample collecting, is highly integrated and flexible. In addition, more function is developing by designing new microfluidic chips or adding some new components to the device. Therefore, this device can be a valuable tool for cell research in space.

1.2 Lab-on-a-chip and its Application in Spaceflight Experiment

Astronauts face serious health threats during spaceflight. The two major factors that may lead to an astronauts’ physiological dysfunction are space radiation and microgravity[2]. Heavy ion radiation, one of the important components of charged particles in orbit, can cause Deoxyribonucleic Acid (DNA) damage and gene mutations. Microgravity can also affect a series of cell physiology functions, including cytoskeleton remodeling, DNA modification, interactions between molecules,. Several ionizing radiation experiments performed in many previous studies suggested the variation in the mutation across different selected immune genes. Scientists performed a further experiment (Immune Gene Mutation-Beijing Institute of Technology-1, IGM-BIT-1) on board the International Space Station (ISS) to explore the molecule evolution rules of the selected DNA. To conduct the on-orbit amplification of the DNA fragments from the antibody encoding genes in the ISS, a self-developed portable and programmable PCR device was designed and produced[3]. They developed a novel PCR chip that consisted of a multi-channel optical adhesive reaction chamber and a miniature thermal cycler. The reaction chamber was cost effective and disposable. The thermal cycler was used to achieve both rapid heating and cooling. As the DNA amplification yield of IGM-BIT-1 PCR device was much similar or even higher than the commercial devices, the IGM-BIT-1 payload has been proven to be suitable for space life science research.

1.3 Design and Surface Modification of a Microfluidic Chip for Intercellular Interactions Research during Spaceflight

Intercellular interactions widely exist in multicellular organisms. The exposure of astronaut’s body to space environment results in a series of biological effects including intercellular interactions. However, these interactions have not been studied extensively in space because of the difficulties faced in performing such experiments in the space. To solve this problem, a co-culture microfluidic chip was designed for studying intercellular interactions and provides an effectively dynamic co-culture method to both adherent cells and suspension cells. Its structure consists of two cell chambers which are divided by polycarbonate semipermeable membrane. The membrane is permeable to signal molecules which are secreted by cells but it is impermeable to the cells itself. Each cell chamber is divided by bolting silk. This results in a control of flow shear stress exerted on the cells and it also results in trapping the suspended cells. As the surface property of the base of any microfluidic chip is important, therefore, they optimized a surface modification strategy using MTS assays and water contact angle test. The results showed that the optimum surface modification strategy is using air plasma treated polystyrene surface for 90 s. Moreover, the contact angle recovery after plasma treatment indicated that the co-culture microfluidic chip should be seeded within 6 days after surface modification. This co-culture microfluidic chip can be a valuable tool for investigating intercellular interactions in space as it can be operated automatically during a spaceflight[1].

2 Biological Study in the Ground-based Simulated Microgravity

2.1 Microbe Research in Simulated Microgravity Condition

2.1.1 Microbiomes of China’s Space Station during Assembly, Integration and Test Operations

Sufficient evidence indicates that orbiting space stations contain diverse microbial populations, which may threaten astronaut health and equipment reliability. Understanding the composition of microbial communities in space stations will facilitate further development of targeted biological safety prevention and maintenance practices. Therefore, this study systematically investigated the microbial community of China’s Space Station (CSS)[4]. Air and surface samples from 46 sites on the CSS and Assembly Integration and Test (AIT) center were collected, from which 40 bacteria strains were isolated and identified. Most isolates were cold and desiccation- resistant and adapted to oligotrophic conditions. Bacillus was the dominant bacterial genus detected by both cultivation-based and Illumina MiSeq amplicon sequencing methods. Microbial contamination on the CSS was correlated with encapsulation staff activities. Analysis by spread plate and qPCR revealed that the CSS surface contained 22.4~54.7 CFU·cm–2culturable bacteria and 9.32×103~ 5.64×10416S rRNA gene copies cm–2; BacLight? analysis revealed that the viable/total bacterial cell ratio was 1.98%~13.28%. This is the first study to provide important systematic insights into the microbiome of the CSS during assembly that describes the pre-launch microbial diversity of the space station. Our findings revealed the following results: (i) bacillus strains and staff activities should be considered major concerns for future biological safety; (ii) autotrophic and multi-resistant microbial communities were widespread in the AIT environment. Although harsh cleaning methods reduced the number of microorganisms, stress-resistant strains were not completely removed; (iii) sampling, storage, and analytical methods for the space station were thoroughly optimized, and are expected to be applicable to low-biomass environments in general. Microbiology-related future works will follow up to comprehensively understand the changing characteristics of microbial communities in CSS.

2.1.2 Fungi from Low-biomass Spacecraft Assembly Clean Room Aerosols

Highly sensitive and rapid detection of airborne fungi in space stations is essential to ensure disease prevention and equipment safety. In this study, quantitative Loop-Mediated Isothermal Amplification (qLAMP) was used to detect fungi in the aerosol of the low-biomass environment of China’s Space Station Assembly Clean room (CSSAC)[5]. A qLAMP primer set for detecting a wide range of aerosol fungi was developed by aligning 34 sequences of isolated fungal species and 17 space station aerosol-related fungal species. Optimization of sample pretreatment conditions of the LAMP reaction increased the quantitative results by 1.29~1.96 times. The results showed that our qLAMP system had high amplification specificity for fungi, with a quantifiable detection limit as low as 102. The detected fungal biomass in the aerosol of CSSAC was 9.59×102~ 2.20×10528S rRNA gene copy numbers m–3. This qLAMP assay may therefore replace the traditional colony-forming unit and quantitative PCR methods as an effective strategy for detecting fungi in space stations.

2.2 Biological Effects in Simulated Microgravity Conditions

To explore the dynamic impacts of Simulated Microgravity (SM) on different vital brain regions of rats, microgravity was simulated for 7 and 21-day, respectively, using the tail-suspension rat model[6,7]. Histomorphology, oxidative stress, inflammatory cytokines, and the expression of some key proteins were determined in hippocampus, cerebral cortex, and striatum. The results showed that 21-day SM decreased brain derived neurotrophic factor and induced neuron atrophy in the cerebral cortex. Strong oxidative stress was triggered at Day 7 and the oxidative status returned to the physiological level at Day 21. Inflammatory cytokines were gradually suppressed and in striatum, the suppression was regulated partially through c-Jun/c-Fos. All of these data revealed that the significant impacts of SM on rat brain tissue depended on durations and regions, which might help to understand the health risk and to prevent brain damage for astronauts in space travel.

Moreover, a proteomic approach was used to investigate rat intestinal homeostasis alterations induced by 7-day simulated microgravity effect[8]. Tail-suspension model was used to simulate microgravity effect and a label-free quantitative proteomic strategy was employed to determine proteins in rat intestine. As a result, 717 differently expressed proteins were identified and 29 proteins were down- regulated while 688 proteins were up-regulated. The three highest enrichment scores were annotation cluster I about cell-cell adhesion, annotation cluster II about carbohydrate metabolism, and annotation cluster III about the activity of peptidase. Results of rat intestine proteomics indicate that SMG might disrupt intestinal homeostasis, which possibly resulted in the opening of Intestinal Epithelial Barrier (IEB), potentially leading to the risk of Systemic Inflammatory Response (SIR) and Inflammatory Bowel Diseases (IBD). The present results also provide some useful information for mechanisms and countermeasures of intestine injuries induced by microgravity.

3 Basic Research for Underlying Mechanisms of Radiation Injury

3.1 Biology Effect of Irradiation on Neuron Cell or Tissue

3.1.1 Autophagy Protects Neural Cells from Carbon-ion Beam Irradiation Injury

The present study investigated autophagy changes and the expression of HMGB1 in human glioblastoma cells[9], responding to carbon-ion beam irradiation (35 keV·μm–1, 80.55 MeV per unit nucleon). U251 cells were irradiated with carbon ion beams and cell proliferation was measured by counting the number of living cells. The expression of Light Chain 3 Beta (LC3B), Beclin 1, High-Mobility-Group Box 1 (HMGB1), pro-form caspase-3, and Cellular FLICE- like Inhibitory Protein (c-FLIP) was analyzed by western blotting. Caspase enzyme activity was determineda caspase cleavage based florescent substrate commercial Kit. Living cell counting demonstrated a time and dose dependent cell death in U251 cells. The expression of LC3B and Beclin 1 revealed that, a high level of autophagy was induced 24 h after irradiation with 1 Gy carbon ions and then decreased in a time- and dose-dependent manner. The expression of the whole HMGB1 showed a good correlation with the dynamic autophagic level. Cytoplasmic HMGB1 maintained autophagy was concluded. Enzyme-Linked Immuno Sorbent Assay (ELISA) measurement found that HMGB1 was released into the extracellular space in a time- and dose-dependent manner. Lower intracellular HMGB1 levels correlated with decreased autophagy as measured by the expression of LC3B. Decreased expression of pro-form caspase-3 and c-FLIP as well as the increased caspase enzyme activity indicated that apoptosis was induced by carbon-ion beam irradiation. Inhibition of HMGB1 release from the area of intracellular to that of extracellular significantly increased cell survival. In summary, carbon- ion beam irradiation could elevate autophagy and HMGB1 expression efficiently, which would protect the cells from programmed cell death via inducing autophagy. Apoptosis as measured by expression of caspase activities increased as the dose increased, which was accompanied with decreased levels of LC3B and HMGB1.

3.1.2 Autophagy Can Promote Apoptosis after Irradiation

As a cancer treatment strategy, irradiation therapy is widely used that can cause DNA breakage and increase free radicals, which leads to different types of cell death. Among them, apoptosis and autophagy are the most important and the most studied cell death processes. Although the exploration of the relationship between apoptosis and autophagy has been a major area of focus, still the molecular me-c-hanisms of autophagy on apoptosis remain unclear. The recent results have revealed that apoptosis was enhanced by the Death Receptor 5 (DR5) pathway, and the effect of autophagy on apoptosis was promoted by DR5 interacting with LC3B as well as Caspase8 in gliomas after irradiation[10]. Interestingly, they observed that the addition of four different autophagy inducers, Rapamycin (RAP), CCI779, ABT737, and Temozolomide (TMZ), induced the differences of DR5 expression and cell apoptosis after irradiation. Unlike RAP and CCI779, ABT737 and TMZ were able to increase DR5 expression and further induce cell death. Therefore, they have concluded that DR5 plays a novel and indispensable role in promoting cell apoptosis under irradiation.

3.2 Carbon Ion Radiation Induced Lipid Disturbance in Brain

The concentration of lipids in the Central Nervous System (CNS) was second only to adipose tissue. Phospholipids (PLs) accounted for 45% of the total brain dry weight and nearly 60% of myelin. The structural unit of lipids was ketoethyl and isoprene, which had both hydrophilic and lipophilic properties and played an important role in cellular structure and function. Lipids also provided energy for the metabolic processes of life, form biofilms, and established an appropriate hydrophobic environment, forming ion channels together with proteins. On the other hand, lipids can also serve as second messengers for signal transduction. Although some studies had shown that space particle radiation causes metabolic disorders of the brain tissue metabolites including 4-butylamic acid, glutamic acid, lactic acid, and other important neurotransmitters[11-15], there was still a lack of in-depth research on the molecular mechanism of brain lipid damage and its relationship with nerve function damage under spatial radiation environment.

In these recent studies, they used UPLC-MS untargeted and targeted lipidomics to screen potential lipid biomarkers in the rat brain induced by simulated space radiation environment from the perspective of lipid molecule changes and to reveal the molecular mechanism of carbon ion radiation-induced brain damage. The 7 weeks male Wistar wild-type rats were exposed to a single high dose of 15 Gy 12C6+radiation vertically on the back of the head at Wuwei Heavy Ion Hospital in Gansu province, China. The control group was consistent with the irradiated group except for not receiving irradiation. UPLC-MS untargeted and targeted lipidomics were performed on the brain tissue and plasma of rats on the seventh-day post-irradiation. Statistical methods such as unsupervised Principal Component Analysis (PCA), supervised Partial Least Squares Discriminant Analysis (PLS-DA) and Orthogonal Partial Least Squares Discriminant Analysis (OPLS-DA) showed that there was a difference in the lipid metabolism in the brain tissue of the irradiated and control groups. In untargeted lipidomics, we screened out 29 differential lipids between the control group and the irradiated group using a multiple test Fold Change (FC)>1.5, PLS-DA model with a VIP>1 and a T test with a<0.1 by volcano plot model. These 29 differential lipids included Diacylglycerol (DAG), Triacylglycerol (TAG), Lysophosphatidylethanolamine (LPE), Lysophosphatidylcholine (LPC), Phos-phatidyl-choline (PC), Phosphatidyleth-anolamine (PE), Sphingomyelin (SM) and branched Fatty Acid esters of Hydroxy Fatty Acids (FAHFAs). The results after classification showed that DAGs were significantly different lipids upon 12C6+radiation. Targeted quantitative analysis of DAGs that had significant changes in the brain tissue and plasma of the irradiated and control rats were also performed to screen out the differential lipids. Based on the quantitative analysis results, they finally screened out three DAG lipids as potential biomarkers for brain damage induced by 12C6+ion irradiation. Interestingly, the contents of these three DAGs in the rat brain tissue and plasma were negatively correlated. The mechanism through which differential lipids induced CNS damage are under explored in our group.

3.3 Bystander Effect Induced by DifferentIrradiation

3.3.1 Mechanism of Bystander Effect Induced by Different Irradiation

Cells exposed with irradiation can induce different biological effects in non-irradiated cells due to the cell-cell interactions. Herein, they investigated the bystander effect of different types of irradiation including Gamma Irradiation (GR) and lithium heavy ion irradiation (LR) on the model human neuroblastoma cell line (SH-SY5Y). The gamma and lithium ion irradiation induced different bystander effects on the SH-SY5Y cell line[16]. The bystander effect induced by gamma irradiation promoted cell proliferation through activating the ERK and AKT signaling pathways, but it could slightly influence the cell cycle of non-irradiated SH-SY5Y cells. Whereas, the bystander effect induced by lithium heavy ion irradiation inhibited the cell proliferation, arrested the cell cycle, and activated the process of pro- apoptosis. The findings of this study confirm the diversified bystander effects of various irradiation on the non-irradiated cells, therefore it highlights the importance of a revised strategy for radioprotection to reduce the damages caused by bystander effects. Further, the in-depth mechanism research on cell proliferation influenced by the bystander effect of radiation will also be useful to understand the biological effects of radiation.

3.4 DNA Mismatching during PCR Reaction Exposed to Space Environment

Mutation is the most important driving force of genome evolution. The pattern of mutation is the key to many questions in evolutional biology. Mutation could be induced by space radiation and microgravity, which are the two major factors leading to astronauts’ physiological dysfunction. Especially the heavy ion radiation, which is one of the important components of charged particles in orbit, can cause DNA damages and gene mutation. Several ionizing radiation experiments performed in our previous study suggest the variation in the mutation across different selected immune genes. Here, we performed a further experiment to explore the molecule evolution of the DNA on the International Space Station (ISS) utili-zing the space environment, providing basic research for life science theory for universe evolution and deep space exploration life science research. This project is the first time an ISS experiment has been independently designed and fabricated in China. It has broken through the bottleneck of Sino-US cooperation in the 30 years, with the meaning of ice- breaking.

To investigate the radiation cumulative effect, two batches of DNA amplification experiments were performed respectively during the spaceflight. The first run was started at the beginning of the on-orbit flight (7–8 June 2017) and the second run was started 10 days after the first one (19–20 June 2017). On 3 July, after the payload returned to earth, the process data and bio-samples are both checked to be good. All samples were analyzed by Agilent 2100 bioanalyzer and the results showed that all the genes were amplified successfully in the space environment.

Some samples were also analyzed by deep sequencing on the HiSeq2500. Sequencing reads were processed for quality control and aligning. We achieved high quality data, and the times of the median depth for all samples was over 8000. For the gene which was composed of the Homosapien immunoglobulin heavy chain variable region germline gene fragments V7~18, D3~10, J2, sequencing data showed there were heteroplasmy both in the ground and space. For the space samples, the average mismatch rate is 1.51% (1st batch) and 1.53% (2nd batch). For the ground samples, the average mismatch rate is 2.67% (1st batch) and 2.74% (2nd batch). However, as there were inevitable errors in the sample preparation and sequencing for the next generation sequencing, ranging from 0.1% to 1%. Ultrasensitive detection of rare mutation and computational analysis of the mismatching information should be further applied in this space experiment samples.

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MA Hong, CHEN Yu, REN Hao, LI Xiaoqiong, YANG Chunhua, LI Bo, HAN Chu, ZHANG Ying, LONG Mian, ZHUANG Fengyuan, DENG Yulin. Space Life Science of China., 2020, 40(5): 928-934. DOI:10.11728/cjss2020.05.928

* Supported by Space Medical Experiment Project of China Manned Space Program (HYZHXM02003)

June 28, 2020

E-mail: deng@bit.edu.cn