Lung adenocarcinoma cells on glutathione endocytosis of nano-Fe3O4

Key words human lung adenocarcinoma cells; glutathione; Fe3O4; endocytosis

Abstract: Objective To observe the in vitro human lung adenocarcinoma cells (SPC-A1) on glutathione (GSSG) Fe3O4 nanoparticles modified with endocytosis and with incubation temperature, time and concentration. Methods Transmission electron microscopy (TEM) and Prussian blue staining cells SPC-A1 Fe3O4 nanoparticles modified GSSG endocytosis. By atomic absorption spectrometry (AAS) method is the amount of SPC-A1 cell adhesion and endocytosis amount of quantitative analysis. Results TEM results showed that cultured 24h at 4 ℃ after the GSSG-modified Fe3O4 nanoparticles adsorbed on the SPC-A1 cells; 37 ℃ 1h cultured cells SPC-A1 after the endocytosis of modified nano-Fe3O4 GSSG significantly increased the number of cells. AAS analysis showed that, SPC-A1 cell surface adhesion amount of Fe3O4 nanoparticles with the incubation time and concentration were positively correlated. GSSG Fe3O4 nanoparticles modified with low concentrations (01 or 04mg/ml), the endocytic capacity in saturated after 24h; GSSG Fe3O4 nanoparticles modified with higher concentration (07 or 10mg/ml), the volume of endocytosis reached after 72h saturated, the largest single cell up to the endocytic 160pg. Conclusion SPC-A1 cells to GSSG Fe3O4 nanoparticles modified endocytosis requires energy, endocytosis is dependent on concentration and incubation time.

Key words: Human lung adenocarcinoma cells; glutathione; Fe3O4; endocytosis

Endocytosis of lung adenocarcinoma cell on glutathione modified magnetite (Fe3O4) nanoparticles
YAN Zhubing, MA Yongjie, GU Hongchen, et al.

Institute of Micro and Nano Science and Technology, Shanghai Jiaotong University (Shanghai 200030, China)

Abstract: Objective To study the relationship between endocytosis of human lung adenocarcinoma cell line SPC-A1 modified by oxidized Glutathione (GSSG) and culture conditions including culture temperature, time and concentration.Methods The uptake of GSSG-modified Fe3O4 nanoparticles by SPC-A1 cell was observed by transmission electron microscopy (TEM) and Prussian blue staining.The extracellular adhesion or intracellular uptake of GSSG-modified Fe3O4 nanoparticles were measured by atom absorb spectrum (AAS). Results TEM results indicated that GSSG-modified Fe3O4 nanoparticles adhered on the membrane of SPC-A1 cell after bEing cultured at 4 ℃ for 1h or 24h, but SPC-Al cell could took up nanoparticles particles after cultured at 37 ℃ for 1h.Prussian blue staining indicated that SPC-Al cells containing GSSG-modified Fe3O4 nanoparticles were much more with the prolongation of culture time or the incre (Chinese papers Union www.lwlm.com order) ase of concentration.AAS analysis indicated that the extracellular adhesion of GSSG-modified magnetic particles was depended on culture time and concentration.The uptake amount of GSSG-modiied Fe3O4 nanoparticles by SPC-A1 cell was saturated after 24h when the nanoparticles in low concentration (0.1mg/ml or 0.4mg/ml), the uptake was saturated after 72h when the nanoparticles in high concentration (0.7mg/ml or 1.0mg/ml). The maximal amount per SPC-A1 cell could reach 160pg.Conclusion The uptake of GSSG-modified Fe3O4 nanoparticles by SPC-A1 cell is energy needed.It depends on the concentration of nanoparticles and saturates at certain time.

Key words: human lung adenocarcinoma cell; glutathione; magenetite (Fe3O4); endocytosis

Oxidized glutathione (GSSG) by the reduced glutathione (GSH) from oxidation, which is widely distributed in 人体各器官 inside. In vivo, GSSG reductase and GSH in the role of the state can be transformed into each other 〔1,2〕 balance, which Fe3O4 nanoparticles modified by GSSG can improve its biocompatibility. Cell endocytosis Fe3O4 nanoparticles can affect the amount of intracellular hyperthermia 〔3〕, magnetic resonance imaging results 〔4〕. In this paper, transmission electron microscopy, Prussian blue staining, atomic absorption spectrometry method for qualitative or quantitative observation of human lung adenocarcinoma SPC-A1 cells to GSSG Fe3O4 nanoparticles modified with endocytosis and with incubation temperature, time, concentration of each other.

1 Materials and methods

11 Synthesis of Fe3O4 magnetic nanoparticles GSSG surface modified nanoparticles: weighing 705g FeCl3 · 6H2O and 297g FeCl2 · H2O, stirred on the magnetic stirrer. Temperature rises to 80 ℃, add 20ml concentrated ammonia, mechanical stirrer reaction 30min, adding 02gGSSG (Shanghai Chemical Reagent Company), under the conditions of reaction at 85 ℃ 1h, remove ammonia, the resultant separated with a permanent magnet repeatedly washed with deionized water 5 times, a steady GSSG modified nano Fe3O4, the concentration of 52mg/ml.

12 GSSG characterization of Fe3O4 nanoparticles modified transmission electron microscope (Philips-TECNAI20S-TWIN)-generated GSSG observed Fe3O4 nanoparticles modified morphology, X-ray diffraction (Bruker-AXS X-ray rotating anode powder diffractometer) measurements its crystal structure.

TEM 13 SPC-A1 cells to GSSG Fe3O4 nanoparticles modified endocytosis of human lung adenocarcinoma cells, Institute of Cell Biology, Chinese Academy of Sciences) were cultured in the recovery of 10% fetal calf serum containing RPMI-1640 medium (American Haike Long Company), placed CO2 incubator (Thermo Corporation) and maintained in culture 37 ℃, 5% CO2. After 24h culture medium replaced with 02mg/ml GSSG Fe3O4 nanoparticles modified culture medium, the growth status of the same dish 3 SPC-A1 cells were cultured according to the following conditions continue: 4 ℃ cultured 1h, 37 ℃ incubation 1h, 4 ℃ incubated 24h. Were collected three kinds of cell culture conditions, using the literature method of sample preparation 〔5〕 observed.

14 GSSG Fe3O4 nanoparticles modified in SPC-A1 cell surface adhesion determination of the amount of SPC-A1 cells to 5 × 105 per well the number of seeded in 6-well culture plates, culture medium volume was 2ml. Cultured at 37 ℃ for 24h, remove the culture medium, the replacement for the 02mg / m.
GSSG Fe3O4 nanoparticles modified culture medium, were cultured at 4 ℃ after the determination of GSSG 0,3,6,12,24 h Fe3O4 nanoparticles modified SPC-A1 cells in the amount of surface adhesion. Also set the concentration of nano-Fe3O4 01 and 04mg/ml, measured the amount of adhesion after 24h culture. Determination by reference 〔6〕.

15 Prussian blue staining at 37 ℃, in the exponential phase of the SPC-A1 cells and 02mg/mlGSSG Fe3O4 nanoparticles modified the culture medium followed by 12 ~ 24h, using Prussian blue staining cells Fe3 + 〔7〕. In addition to set 01 or nano-Fe3O4 concentration of 10mg/ml, Prussian culture stained after 24h. Chinese papers Alliance WWW.LWLM.COM finishing.

16 SPC-A1 cells to GSSG Fe3O4 nanoparticles modified endocytic contents at 37 ℃, SPC-A1 cells to 5 × 105 per well the number of seeded in 6-well culture plates, culture medium volume was 2ml. After 24h culture, the culture medium replaced by Fe3O4 nanoparticles modified with GSSG in the culture medium. Concentration of nano-Fe3O4 were 05,1,15,20 mg / ml. Four kinds of concentration were cultured 0,3,6,12,24,48,72 h, determination of SPC-A1 cells to GSSG Fe3O4 nanoparticles modified endocytic capacity. Determination by reference 〔6〕.

2 Results

21 GSSG characterization of Fe3O4 nanoparticles modified (Figure 1) transmission electron microscope, GSSG Fe3O4 nanoparticles modified for the uniform spherical particles with narrow size distribution, particle size is 7 ~ 15nm. GSSG-modified nano-Fe3O4 magnetic fluid formed in aqueous solution, ultrasonic treatment has a good dispersion and stability. XRD spectra of the samples that the samples are cubic spinel structure.

Figure 1 GSSG Fe3O4 nanoparticles modified TEM image (scale to 20nm) (omitted)

22 transmission electron microscope at 4 ℃ SPC-Al cells and 02mg/mlGSSG Fe3O4 nanoparticles modified the culture medium after 1h, nano Fe3O4 SPC-A1 cell adhesion to the surface is not internalized into the SPC-Al cells (Fig. 2a), After 1h at 37 ℃ culture GSSG Fe3O4 nanoparticles modified to internalize into SPC-A1 cells (Fig. 2b), incubated at 4 ℃ for 24h, nano Fe3O4 still not internalized into SPC-A1 cells (Fig. 2c).

Note: "" indicates that GSSG-modified magnetic nanoparticles; a: 4 ℃ after 1h culture TEM image; b: 37 ℃ after 1h incubation TEM image; c: 4 ℃ after 24h incubation TEM images

Under different culture conditions Figure 2 SPC-A1 cells by transmission electron microscopy images (omitted)

23 GSSG Fe3O4 nanoparticles modified in the SPC-Al cell surface adhesion amount of GSSG Fe3O4 nanoparticles modified in SPC-A1 cell surface adhesion increased with the prolongation of culture time increased, GSSG Fe3O4 nanoparticles modified cells in SPC-A1 Adhesion is dependent on GSSG concentration of Fe3O4 nanoparticles modified, both showed a linear relationship.

24 Prussian blue staining when the culture medium GSSG Fe3O4 nanoparticles modified when the concentration of 02mg/ml, endocytosis after 24h culture GSSG Fe3O4 nanoparticles modified the SPC-A1 was more than the number of cultured cells after 12h (Figure 3a). When the incubation time was 24h, with medium to increase the concentration of nano-Fe3O4, endocytic GSSG Fe3O4 nanoparticles modified by the number of SPC-A1 cells was significantly increased (Figure 3b, Figure 3c, Figure 3d)

25 SPC-A1 cells to GSSG Fe3O4 nanoparticles modified endocytic capacity (Chinese papers Union www.lwlm.com order) SPC-A1 cells to GSSG Fe3O4 nanoparticles modified endocytosis is dependent on GSSG concentration of Fe3O4 nanoparticles modified, Fe3O4 nanoparticles modified with the GSSG concentration corresponding endocytosis also increased. Fe3O4 nanoparticles modified when the GSSG concentration of 01 and 04mg/ml, the cultured cells SPC-A1 after 24h endocytosis reached saturation; Fe3O4 nanoparticles modified when the GSSG concentration of 07 and 10mg/ml, the SPC-A1 after 72h culture endocytic cells reached saturation. SPC-A1 cells to a single GSSG Fe3O4 nanoparticles modified up to the endocytic capacity 160pg.

Note: a: GSSG concentration of Fe3O4 nanoparticles modified 02mg/ml, incubation time 12h;

b: GSSG concentration of Fe3O4 nanoparticles modified 02mg/ml, incubation time 24h;

c: GSSG concentration of Fe3O4 nanoparticles modified 01mg/ml, incubation time 24h;

d: GSSG concentration of Fe3O4 nanoparticles modified 10mg/ml, incubation time 24h

Figure 3 Prussian blue staining images (200 ×) (omitted)

3 Discussion

In this paper, transmission electron microscopy at 4 ℃ for 1 or 24h after training, GSSG Fe3O4 nanoparticles modified only SPC-A1 cell adhesion to the surface, not internalized into the cells. And 37 ℃ for 1h after training, SPC-A1 cells are able to internalize the GSSG-modified nano-Fe3O4. SPC-A1 cells confirmed that GSSG Fe3O4 nanoparticles modified without the energy of adhesion, the role of internalization requires energy. This paper studies the impact of GSSG at 4 ℃ Fe3O4 nanoparticles modified SPC-A1 cell adhesion to the surface of the relevant factors. The results showed that the adhesion amount of time and with the culture medium Fe3O4 nanoparticles modified with the concentration of GSSG showed a linear relationship. Many foreign scholars have studied different cell Fe3O4 nanoparticles of different endocytosis. The results show that cells of Fe3O4 nanoparticle endocytosis and cell types, Fe3O4 nanoparticles surface modification, size _ the culture time, Fe3O4 nanoparticles concentration, temperature and other factors 〔8,9〕. This Prussian blue staining results showed that with prolonged incubation time or increasing the concentration of Fe3O4 nanoparticles in SPC-A1 significantly increased the number of cells, internalization of the GSSG-modified nano-Fe3O4 are all located in the cytoplasm. AAS results show that Fe3O4 nanoparticles modified with the concentration of GSSG increased iron content in individual cells increased, the reason may be to increase the concentration of nano-particles, nano-particles increased the adhesion of nanoparticles led to an increase in internalization. SPC-A1 cells to GSSG Fe3O4 nanoparticles modified endocytic capacity will be saturated with culture time and cell growth may be a single cellular iron status and on the capacity of saturation.

References


〔1〕 Jordan A, Scholz R, Klaus MH, et al.Presentation of a new magnetic field therapy system for the treatment of human solid tumors with magnetic fluid hyperthermia [J]. Journal of Magnetism and Magnetic Materials, 2001,225:118

Medicated bath in the prevention of disease and application of the mechanism

Abstract: This paper discusses the mechanism of strong medicine bath, introduces bath and Zhuang medicine commonly used features of drugs commonly used to explain Zhuang medicine bath application in the prevention of disease.

Keywords: strong medicine bath; mechanism; Application

Medicated bath treatment outside the traditional law of the motherland, the people of ancient China and the physicians working in the struggle with the disease process of wisdom. Archaeological data show that strong medicine bath has a long history, much in the Stone Age Zhuang ancestors learned that is budding time of the fire, the Zhuang people for thousands of years which an effective means of preventing and curing diseases and methods. Since ancient times, medicine bath Zhuang Zhuang people has been the prevention of disease and physical health of the important ways, especially in recent years, as people's living standard continues to improve, people in traditional medicine, especially in the advocacy of national medicine is becoming more , the bath and other ethnic groups, including Zhuang medicine preventive medicine methods of continuous attention to disease prevention Zhuang medicine becoming more and more attention, Zhuang Zhuang medicine bath in very rapid development momentum in the region. Therefore, this strong medicine to bath in the prevention of disease mechanism and application to talk about their Chuqian view.

A strong mechanism of Medicine bath

Zhuang Zhuang Medicine Hospital in Bath in the prevention of diseases, to the unique strong medical theory, combined with long experience in the formation of the transmission, prevention, health care, health, disease treatment and rehabilitation have good results. Medicated bath drug selection is relatively simple, according to Zhuang medicine theory, individual, season of the different selection of various drugs, through seepage bubble, cooking, using heat, or heat with the drug, acting on the body of the body or certain parts of Long Road by clear, Fire Road, the blood, regulate day, people, places three simultaneous gas balance, so as to achieve temperature by dispelling, qi and blood circulation, Shu qi, Yishenqiangxin waist, detoxification, swelling and pain, to reconcile yin and yang, coordinating the internal organs, body care solution and other effects, and ultimately disease prevention, physical fitness purposes.

Modern medicine, drugs and medicated bath of hot water through the role of the physical heat, skin telangiectasia, blood flow to accelerate, promote blood circulation, increase metabolism and disease resistance, clear metabolites accelerate the pathology, the whole body tissues and organs improve the nutritional status and reduce the excitability of nerve endings and a muscle relaxation effect, help eliminate fatigue, promote sleep, the normal health care and rehabilitation of sick body have good effect.

2 common bath and features strong medicine

Medicated bath method, many different types of drugs into water bath and steam bath medicine, drugs are divided into natural mineral water bath water bath and warm bath Zhuang medicine; in accordance with the role of the body Author: Yu-blue camp, 1968 -, male, associate professor engaged in medical and Zhuang Medicine Teaching and Research. Tel: 13878883950, E - mail: yuyinglan2005 @ 126. Com spot is different, is divided into partial bath bath and body, and to the local baths for the more local bath is divided into hot bath, fumigation, foot bath, bath and so on. Medicated bath therapy, many different types are several commonly used method is.

2.1 bath natural drug. Natural medicated bath is more important non-significant one of the hot dipping, which was mainly due to the rain through the floor, including Zhuang medicine, including herbal plant litter soaking and filtering out the infiltration of ground water flow along the valley to form a rich variety of medicinal plants in natural herbal spa ingredients, this natural herbal medicine bath water content is very high, with beauty, bleaching, sterilization, the role of physical health, alleviate foot fatigue for health especially. This medicine bath without special processing production, is natural.

2.2 Medicated washes therapy. Yuzu is a strong medicine for the disease one of the methods commonly used, has a long history. Yuzu is to add boiling herbs for 30 minutes, filter, when the temperature dropped to 40 ~ 50 ℃, the foam is used to wash the feet or foot. Yuzu has pass Lung Road, Fire Road qi, detoxification, anti-inflammatory pain, swelling and blood stasis, pesticides and other effects itching, skin heat evenly, Cou clear, blood vessels expand, blood flow, to achieve the prevention of disease purposes.

2.3 Zhuang radiopharmaceuticals hot iron bath. Is the use of the drug and heat to prevent and treat diseases of the important methods. People often folk intense fragrance, take the channeling of the drug as a stimulus to iron therapy drugs; Zhuang medicine hot iron therapy is the use of heat, or heat with the drug, ironing certain parts of the body to clear the long road, fire road blood, regulate days, the people, to synchronize the three gas balance, so as to achieve an external treatment for therapeutic purposes. According to non-drug and drug use in the human body is divided into sub-heating non-drug therapy and drug therapy two categories.

2.4 bath fumigation Zhuang radiopharmaceuticals. Zhuang medicine physical therapy is by steam cooking vapors smoked the drug affected area, and to disease prevention and treatment of disease. The commonly used drugs mixed pounded dried meal, put empty barrels, or crypt in burning, smoke and take the heat so bake smoked affected area. Or are based on purpose, use of different drugs head, face or body Jiantang fumigation. This approach medication, relatively few contraindications, whichever is more than medicine and Lee Hom, running blood, the provision of foul Chubing, so the indications of this Law is very broad. 3 bath Zhuang medicine commonly used drugs

Bath features a strong medicine to prevent disease, according to no purpose without the drugs selected to achieve different results. Commonly used drugs according to different conditions may be. The prevention of rheumatic joint pain, low back pain and other pain syndrome, often used in HE, Ling Xian, through sound casual, Pittosporum skin, camphor grass, two needles, pomelo leaves, citrus fruit leaves, large Luo umbrella, Ronaldinho umbrella, wide bars vine, ivy, wind Ai, cinnamon and so on. The prevention of colds, common wind, Nepeta, Guanzhong leaves, cassia twig, chrysanthemum, grass River car, ephedra, Caulis Lonicerae. Prevention of acute eczema and other skin diseases can be Nepeta, wind, gypsum, Sophora, herb, beef sub, raw land, Chan Yi, raw licorice.

4 Zhuang medicine bath application in the prevention of disease

Bath according to the characteristics of strong medicine, strong medicine bath in a wide range of disease prevention, especially prevention of external emotional diseases, skin diseases, wind cold sore disease, fever psychosis.

4.1 Prevention of external emotional disorders. Whenever catch cold by the wet, multi-party Jianshui shower bath with onion ginger to sweat solution form, fend off the cold outside to prevent moisture gain emotional disorders. If the exogenous and internal injuries, resulting in clever dock No Network, caused by the pain the patient conscious head, Zhuang Jian Medical people used the Millennium, Speranskia grass, chasing the wind, a GE of the 10g, gauze, Jianshui shampoo to relieve pain, prevent disease worse.

4.2 prevention of skin infection. Met with a certain infectious skin disease, due to restrictions, the daily use in your face, wash the body of the tool can not be used alone, in order to prevent skin disease transmission to other members of the family, Zhuang Zhuang medicine in some areas would often put in pot boils water to bathe, which can prevent the spread of skin diseases, and this method is still used some of Zhuang Autonomous Region.

4.3 prevention of pain syndrome. Most of the Zhuang people live in the Mid-levels, most of them live in the valley or Pingba the waterfront area, akin to finding far, in their daily activities, including the introduction of a variety of labor, such as the heel is often like the ribs disease, joint pain syndrome occurred. Therefore, Zhuang Zhuang people doctors often prevent foot pain syndrome one of the important ways and means. Zhuang people live and the land of rain, humid, more common in the elderly suffering from rheumatism. Therefore, people often Zhuang Zhuang medicine radiopharmaceuticals iron bath to prevent rheumatic fever from occurring, common iron bath: lycopodii 20g, lost his stick, Mountain King, eighteen patients, independent real trees, red fish eyes, cathayensis The Gui 30g, crushed bag wrapped involved, the first 1500 to 2000 ml of water immersion after 20 minutes, heated fry boil for 15 minutes, the drug bag while still hot (as is appropriate to adapt to the heat) often appear repeatedly pressing Rheumatology place, l 5 min after administration of water bath to wash the affected area.

4.4 to prevent the occurrence of fever disease. Daily sun and rain, sweat but feel cold very easily lead to the occurrence of fever disease. Therefore, the Zhuang people every summer, when toiling encountered rain, most of them with ginger, onion, 50g of each pounded home in the tub, into the water caused Banpen, stir well, so when the appropriate temperature, body scrub, to prevent the occurrence of fever disease.

4.5 Yuzu recover from fatigue Zhumian healthy. Zhuang people have a bubble with hot water before going to sleep habits of the foot, through the use of hot water washes, both warm the body, can relieve fatigue during the day to help sleep, to promote health and longevity. If according to their physical condition, when the water washes the appropriate add some strong medicine. The prevention of disease, treatment of disease can play a good supporting role. Such as adding lycopodii, Zeeland, hematoxylin and other drugs, to relieve fatigue, promote sleep is more beneficial; appropriate to join Gui, ginger, mulberry and other drugs with effects of warming, more conducive to people with rheumatism, for fear cold, stomach Deficiency person; Jiaru Luo Ying wood, Ur, Prunella and other drugs with lower blood pressure, people with high blood pressure may also play a role of adjuvant therapy.

4.6 prevention of neonatal disease. Neonatal health-care bath, pig bile 30ml decoction bath, moisturizing the skin smooth and newborns can not sores ringworm.

4.7 to prevent the occurrence of skin diseases. With Mahonia, nine years out, Vaccaria Hot water washes, day 1, and promote blood circulation to the feet of diabetic patients to prevent metabolic disorders, sugar and protein deposition in blood vessels, causing atherosclerosis and arterial wall stenosis and susceptible to various skin diseases, such as shingles, athlete's foot and so to a certain extent.

In summary, the Zhuang Zhuang medicine medicine theory in the bath under the guidance of the drug on the water, use warm water to stimulate, by soaking, bathing, washing of local or systemic, to achieve the prevention of disease, a preventive health care and physical health methods. As Zhuang medicine bath is very simple to operate, the use of the drug or drugs can make the best of most medicated bath, medicine source is also very extensive and very convenient to use, usually non-toxic side effects, easy to impose on children and even adults, Therefore, we can as a "disease" is an important health care and to promote the use of therapies.