A method for detecting the function of androgen receptor under microgravity conditions

Abstract

The present invention relates to the technical field of detecting androgen receptor function, and discloses a method for detecting androgen receptor function under microgravity conditions, including culturing osteoblasts using microgravity gelatin, and obtaining a mathematical modeling formula for the osteogenic function of AR based on three data: calcium ion concentration and reaction time, the nuclear-cytoplasmic ratio of androgen receptor (AR), and the quantitative expression of osteoblast markers; this method not only establishes a connection between AR and microgravity, but also can quantitatively describe based on mechanical load and bone structure data, realizing the tracking and monitoring of androgen receptor function under microgravity conditions; and it helps to study and clarify the relationship between androgen receptor and bone mechanical remodeling, promoting the development of new anti-osteoporosis drugs that can restore the physiological structure of bone and enhance bone strength at the same time, and has important application value.

Description

Technical Field

[0001] The present invention relates to the technical field of detecting androgen receptor function, and particularly relates to a method for detecting androgen receptor function under microgravity conditions. Background Art

[0002] Under microgravity conditions, bone loss and osteoporosis will occur, and the fundamental cause of bone loss is the decline in the function of osteoblasts.

[0003] Androgen receptor (AR) belongs to the steroid receptor in the nuclear receptor superfamily. AR generally consists of four domains: N-terminal transcriptional activation domain (NTD), DNA binding domain (DBD), hinge region, and ligand binding domain (LBD). It is expressed in various tissues of the human body and is a receptor expressed through the physiological and pathophysiological effects of endogenous androgen ligands such as testosterone (T) and dihydrotestosterone (DHT).

[0004] Previous studies have suggested that the mechanical response of bone remodeling is related to the function of androgen receptor (AR). However, existing experimental methods cannot track and detect the functional changes of androgen receptor in a microgravity environment; therefore, a new method is needed to establish a connection between the mechanical response of bone tissue under microgravity conditions and the functional changes of androgen receptor. Summary of the Invention

[0005] To solve the above technical problems, the present invention provides a method for detecting androgen receptor function under microgravity conditions, including the following steps:

[0006] (1) Osteoblasts are cultured with 3D microgravity gelatin for 21 days;

[0007] (2) Laser confocal microscopy is used to observe the dynamic changes of calcium ion concentration in osteoblasts;

[0008] (3) Western Blot is used to detect the nucleo-cytoplasmic ratio of androgen receptor (AR);

[0009] (4) Cell proteins are extracted for mass spectrometry to detect the expression of osteoblast markers;

[0010] (5) Mathematical modeling is carried out based on the continuous expression of osteoblast markers, calcium ion concentration, and AR nucleo-cytoplasmic ratio for 21 days, and its formula is as follows:

[0011] (6) The osteogenic function of AR under different degrees of microgravity conditions is obtained according to the obtained mathematical model.

[0012] Further, the 3D microgravity gelatin in step (1) is 1 kPa and is prepared from 10 mg / mL Glycosil and 10 mg / mL Gelin-S.

[0013] Further, step (2) also includes applying a stress stimulus before observing the dynamic changes in calcium ion concentration.

[0014] Compared with the prior art, the beneficial effects of the present invention are as follows: This method not only establishes a connection between AR and microgravity, but also can quantitatively describe based on mechanical load and bone structure data, realizing the tracking and monitoring of androgen receptor function under microgravity conditions; and it helps to study and clarify the relationship between androgen receptor and bone mechanical remodeling, promoting the development of new anti-osteoporosis drugs that can restore the physiological structure of bone and enhance bone strength at the same time, having important application value. BRIEF DESCRIPTION OF THE DRAWINGS

[0015] Figure 1 It is the change in calcium ion concentration and reaction time in osteoblasts after cultivation according to the present invention;

[0016] Figure 2 It is the AR content in the cytoplasm and nucleus of osteoblasts after cultivation according to the present invention, where Lamin A / C is the internal reference for nuclear protein; the left side is the cytoplasmic AR content, and the right side is the nuclear AR content. DETAILED DESCRIPTION OF THE EMBODIMENTS

[0017] To make the objectives, technical solutions, and advantages of the present invention clearer and more understandable, the present invention will be further described in detail below in conjunction with the embodiments and the drawings. The illustrative embodiments and descriptions of the present invention are only used to explain the present invention and do not limit the present invention.

[0018] Example:

[0019] Under microgravity conditions, the osteogenic function of AR will be weakened, but it has a non-linear relationship with osteoblast markers; its function is simultaneously affected by calcium ion concentration and reaction time; the cytoplasmic-nuclear ratio of AR; and the microgravity action time and microgravity degree. Therefore, a method for detecting androgen receptor function under microgravity conditions is needed.

[0020] A method for detecting androgen receptor function under microgravity conditions includes the following steps:

[0021] (1) Osteoblasts were cultured with 3D microgravity gelatin to simulate the microgravity environment. The osteoblasts were MC3T3-E1 cells. At the same time, 10-8M testosterone, 50 μg / ml vitamin C, and 10 mM β-glycerophosphate were added. The microgravity gelatin (1 kPa) was prepared from 10 mg / mL Glycosil + 10 mg / mL Gelin-S and cultured for 21 days. Every day during the culture process, the calcium ion concentration and reaction time of osteoblasts were detected; the cytoplasmic-nuclear ratio of androgen receptor (AR); the quantitative expression value of osteoblast markers. The 18th day was selected as the representative data. At this time, the osteoblasts were in the mature stage and were more likely to reflect the phenomenon of AR promoting or weakening the function of osteoblasts under microgravity conditions.

[0022] (2) During the 0-21 days of 3D microgravity gelatin culture, the green fluorescent calcium indicator Fluo-4AM was used for labeling, and an excitation light with a wavelength of 488 nm was given by a laser confocal microscope to observe the dynamic changes of intracellular calcium ion concentration and the reaction time (s) when it reached the peak; as Figure 1 shown (representative data on the 18th day), the calcium ion concentration and reaction time showed a non-linear relationship, showing that the calcium ion concentration first increased and then decreased (the vertical coordinate represents the calcium ion concentration signal after fluorescence excitation, the horizontal coordinate is the reaction time of calcium ion concentration after fluorescence excitation, and the time to reach the peak is used as the denominator to calculate the calcium ion concentration increase rate).

[0023] (3) During the culture process of osteoblasts under microgravity (0-21 days), the cytoplasmic-nuclear ratio of androgen receptor (AR) was detected by Western Blot, as Figure 2 shown (representative data on the 18th day); LaminA / C was used as the internal reference for nuclear proteins. The left side was the content of cytoplasmic AR, and the right side was the content of nuclear AR. The cytoplasmic-nuclear ratio of AR was calculated according to the gray value.

[0024] (4) Cell proteins were extracted for mass spectrometry detection to clarify the quantitative expression of osteoblast markers;

[0025] As shown in Table 1, the main indicators representing osteogenic function were the expressions of osteoblast markers (Osteocalcin is osteocalcin; Osteopontin is osteopontin). The higher the expression, the stronger the osteogenic function of AR. Its accurate quantitative expression can be defined as the Score Sequest HT value of mass spectrometry analysis, and Coverage is the confidence level. A value greater than 10 is considered credible.

[0026] Table 1: Expression of osteoblast markers (on the 18th day)

[0027]

[0028] (5) Mathematical modeling was carried out based on the expression of osteoblast markers, calcium ion concentration, and AR nuclear-cytoplasmic ratio over 21 consecutive days.

[0029] Based on the data of the continuous expression of osteoblast markers, calcium ion concentration / reaction time, and AR nuclear-cytoplasmic ratio during the 0-21-day culture process, a dose-effect relationship formula for the osteogenic function of AR was constructed (using the optimization function solved by Matlab language); quantitative and calibration calculations were performed on the expression level of osteogenic markers representing the osteogenic function of AR to directly reflect the osteogenic function of AR under different microgravity conditions; compared with previous methods, this method quantitatively analyzed the osteogenic function of AR under microgravity for the first time, changing the drawback of only indirectly estimating the function of AR through the expression level of osteogenic markers in the past. When the ratio is greater than 1, it represents a strong osteogenic function of AR; when the ratio is less than 1, it represents a weakened osteogenic function of AR. The formula is as follows:

[0030]

[0031] Note: The stress is less than 40 kPa (simulation of the gravity of physiological bones, the maximum stress is 40 kPa)

[0032] Compared with previous methods, this method quantitatively analyzed the osteogenic function of AR under microgravity for the first time, changing the drawback of only indirectly estimating the function of AR through the expression level of osteogenic markers in the past.

[0033] (6) The osteogenic function of AR under different degrees of microgravity conditions was obtained according to the obtained mathematical model.

[0034] According to this model, the intensity of the osteogenic function of AR under different microgravity conditions can be quantitatively calculated by detecting the expression of osteoblast markers, calcium ion concentration, and reaction time; repeating the above experiments with microgravity gelatin (20 kPa and 39 kPa), and substituting the obtained data into the above calculation formula to obtain the osteogenic function of AR, which proves that this method has high feasibility and accuracy.

[0035] Through the above embodiments of the present invention, the present invention can realize the tracking and monitoring of the function of androgen receptor under microgravity conditions, which is helpful for studying and clarifying the relationship between androgen receptor and bone mechanical remodeling, and promoting the development of drugs that can restore the physiological structure of bone; at the same time, it has important application value for the research and preparation of new anti-osteoporosis drugs that enhance bone strength.

[0036] The above is the embodiment of the present invention. The above embodiments and the specific parameters in the embodiments are only for clearly expressing the invention verification process, and are not used to limit the patent protection scope of the present invention. The patent protection scope of the present invention still depends on its claims. Any equivalent structural changes made by using the content of the specification and drawings of the present invention should also be included in the protection scope of the present invention by the same token.

Claims

1. A method for detecting the function of androgen receptor under microgravity conditions, characterized in that, it comprises the following steps: (1) Osteoblasts are cultured with 3D microgravity gelatin. The osteoblasts are MC3T3-E1 cells. At the same time, 10−8 M testosterone, 50 μg / ml vitamin C and 10 mM β-glycerophosphate are added. The microgravity gelatin (1 kPa) is prepared from 10 mg / mL Glycosil and 10 mg / mL Gelin-S, and cultured for 21 days; (2) Laser confocal microscopy is used to observe the dynamic changes of intracellular calcium ion concentration and the reaction time (s) when it reaches the peak; (3) Western Blot is used to detect the cytoplasmic-nuclear ratio of androgen receptor (AR), where the cytoplasmic-nuclear ratio of AR is the ratio of the content of AR in the cytoplasm to the content of AR in the nucleus; (4) Cell proteins are extracted for mass spectrometry detection to clarify the quantitative expression of osteoblast markers. The quantitative expression of osteoblast markers is the sum of the Score Sequest HT values of the mass spectrometry analysis of osteocalcin and osteopontin; (5) Mathematical modeling is carried out based on the quantitative expression of osteoblast markers for 21 consecutive days, calcium ion concentration signal, reaction time (s) when the calcium ion concentration signal reaches the peak, microgravity time (days), stress (kPa) and AR cytoplasmic-nuclear ratio. The formula is as follows: ； (6) The osteogenic function of AR under different degrees of microgravity conditions is obtained according to the obtained mathematical model.

2. The method for detecting the function of androgen receptor under microgravity conditions according to claim 1, characterized in that, step (2) further includes applying stress stimulation before observing the dynamic changes of calcium ion concentration.

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