CN1384186A

CN1384186A - Biological tissue unicell vibrating sepaation method and separator

Info

Publication number: CN1384186A
Application number: CN 01113590
Authority: CN
Inventors: 徐天乐; 伍龙军; 孔令芳
Original assignee: University of Science and Technology of China USTC
Current assignee: University of Science and Technology of China USTC
Priority date: 2001-04-27
Filing date: 2001-04-27
Publication date: 2002-12-11

Abstract

The present invention relates to the vibration separating method to separate unicell from biological tissue and the vibration separator. The present invention is characterized by that the vibrating pointer is fixed on the vibrator, and the vibrator consists of regulated power supply, waveform generator, power amplifier and hot pen record. The vibrator is fixed onto the manipulator; and the waveform generator consists of a ICL8038 chip and peripheral circuits. The vibration frequency is changed via regulating the variable capacitor in the peripheral circuits and the vibration amplitude is changed via regulating the potentiometer. After the biological tissue is fixed, the vibration pointer is made to vibrate in 5-10 Hz frequency and 100-200 micron amplitude and separated unicell may be obtained.

Description

Biological tissue unicell vibrating sepaation method and tripping device

The invention belongs to the separation method technical field of biological tissue cell, particularly mechanical separation device.

According to U.S.'s " Journal of Neuroscience " (nineteen eighty-three the 3rd volume 1888-1899 page or leaf), at present, being applied to the drosophila larvae axoneuron, to obtain single celled separation method be by enzymes such as various proteolytic enzyme and collagenases tissue to be carried out enzymolysis, but the enzyme solution of this routine is of a high price and time-consuming.Britain's " nature " magazine (1977 the 265th volume 751-753 pages or leaves) is pointed out, some albumen on enzyme pair cell surfaces such as proteolytic enzyme of using in the enzyme solution and collagenase have certain digestion, thereby make and isolatedly unicellularly can not reflect cell activity under the physiological status well.Britain's " cell cultures progress " (1981 the 1st volume 125-182 pages or leaves) points out, though at present to all systematic research of fruit bat ovum, larva, pupa, yet because the conventional enzyme solution of the unicellular usefulness of adult axoneuron is difficult to acquisition, so do not see the report that the single neurone of fruit bat adult is studied so far as yet.

The present invention proposes a kind of with unicellular vibrations separation method of separating from biological tissue and vibrations tripping device thereof, to overcome the above-mentioned defective of existing enzymolysis separation method.

Fig. 1 is the synoptic diagram of the unicellular vibrations tripping device of biological tissue of the present invention;

Fig. 2 is the schematic circuit of the oscillator in the vibrations tripping device.

The single celled vibrations tripping device of biological tissue of the present invention is characterized in that the upward fixing vibrations pointer (II) that can thereupon shake of earthquake device (I), and oscillator (I) is fixed on the Manipulators (III); Described oscillator (I) is composed in series successively by voltage stabilized source (V), waveform generator (VI), power amplifier (VII) and heat writing recorder (VIII); Wherein waveform generator (VI) is made up of chip IC L8038 and peripheral circuit; The frequency that changes concussion by the resistance that disposes in the peripheral circuit and variable capacity; The waveform that waveform generator (VI) sends links to each other with heat writing recorder (VIII) by a potentiometer W after power amplifier (VII) amplifies, and is used to change the amplitude of concussion.

The present invention is with the unicellular vibrations separation method of separating from biological tissue, it is characterized in that: after required isolating biological tissue is fixed, making the vibrations pointer on the shaking device is that 5-10Hz, amplitude are that 100-200 μ m shakes organizationally with the frequency, thereby obtains isolating unicellular.

Compare with existing enzymolysis separation method,, do not need the effect of enzyme because the present invention's employing is that the vibrations separation method is a mechanical separation method, thus the time and efforts of the required cost of enzymolysis can be saved, both very economical, convenient and swift again; What is more important, mechanical separation method have overcome enzymolysis and have brought the cell disadvantageous effect that digestion own causes, make isolated unicellular more approaching at body physiological status and have higher activity.

The present invention shakes the unicellular separation that separation method and device can be used for various biological tissues.Particularly, the contriver utilizes present method and device thereof to isolate the axoneuron of the fruit bat adult that forefathers never separated; By electrophysiology and pharmacological evaluation, prove that these fruit bat axoneurons have good physiologically active.This is to be difficult to obtain with conventional enzyme solution.

Below in conjunction with description of drawings the specific embodiment of the present invention.

Fig. 1 is the vibrations tripping device synoptic diagram that is used to separate the fruit bat axoneuron in the embodiment of the invention.

Fig. 2 is the schematic circuit of oscillator in the present embodiment vibrations tripping device.

Fig. 3 is that present embodiment amplifies 600 times phase microscope picture with the isolating fruit bat of vibrating method larva of a tapeworm or the cercaria of a schistosome axoneuron in three ages, and 1 and 2,3 and 4,5 and 6 represent I, II and III type neurone in the fruit bat larva of a tapeworm or the cercaria of a schistosome central nervous system in three ages respectively among Fig. 3.

Among Fig. 41,2,3 and 4 is full cell currents of four types that record from isolating fruit bat larva of a tapeworm or the cercaria of a schistosome II in three ages type neurone.

Among Fig. 51,2 and 3 is respectively fruit bat adult forebrain successively and amplifies 600 times phase microscope picture with isolating two the fruit bat adult axoneurons of vibrating method.

Among Fig. 61,2,3 and 4 is full cell currents of four types that record from isolating fruit bat adult neurone.

Embodiment 1: the vibrations of fruit bat axoneuron separate

One, tripping device

The vibrations tripping device that adopts in the present embodiment, earthquake device I are gone up the fixing vibrations pointer II that can thereupon shake, and oscillator is fixed on the Manipulators III; Oscillator I wherein is composed in series successively by the heat writing recorder VIII that L7809 type voltage stabilized source V, waveform generator VI, TDA2030 type power amplifier VII and Shanghai Medical Equipment Plant's July 1st produce; What the Manipulators III in the present embodiment adopted is the mechanical narishige of MM-3 type that Japan that Rui Shi (Narishige) company produces; Also can select other narishige for use as DYN dynamic, fluid pressure type or piezoelectric ceramic type.Heat stylus recorder VIII is fixed on the narishige III, and the vibrations pointer II of the tip about 5-10 μ m that is drawn into by Glass tubing or glass stick is fixed on the heat stylus recorder VIII; Frequency and amplitude by waveform generator VI control oscillator II; Oscillator I is fixed on the narishige III with rivet.

Oscillator is to utilize heat stylus recorder to trace the principle of waveform, controls the electromechanical device of heat stylus recorder with certain frequency and amplitude vibrations by a waveform generator; Wherein waveform generator VI is made up of chip IC L8038 and peripheral circuit, and from the pin two sine wave output of chip IC L8038, sinusoidal wave frequency is determined by R1 and total capacitance: the resistance R 1 of two 8.2K Ω is connected on respectively between

chip pin

4,5 and the positive source; The capacitor C of 3 μ F is connected between chip pin 10 and the power cathode, connect altogether by the C3 of C2, the 0.4 μ F of C1, the 0.2 μ F of 0.1 μ F and C4 one end of 0.8 μ F and the capacitor C of 3 μ F, the other end composes in parallel the selection capacitor bank by system selector switch K with any one electric capacity in the capacitor bank and capacitor C respectively, be used for changing total capacitance to change frequency, also promptly change the vibration frequency of vibrations pointer; Resistance is that the R3 of 30K Ω and the R4 of 8.2K Ω are used for adjusting sinusoidal wave waveform, one group of R3 is connected between pin one and the positive source, R4 is connected between pin one and the power cathode, and another group R3 is connected between pin one 2 and the power cathode, R4 is connected between pin one 2 and the positive source; Resistance is that the pull-up resistor R2 of 1K Ω is connected between pin 9 and the positive pole; Pin 6 and anodal short circuit;

7 and 8 short circuits; Pin one 1 connects negative pole.Can change sinusoidal wave output amplitude by regulator potentiometer W, to reach the amplitude that changes the vibrations of vibrations pointer.

Two, material and working method:

Sample: drosophila strains is that U.S. Iowa (Iowa) university bio-science is wild-type Canton-S and the Oregon-R strain that professor Wu Chunfang provides.With the fruit bat larva of a tapeworm or the cercaria of a schistosome in three ages with washed with de-ionized water after, be placed on to drip on the slide glass have and dissect, isolate its scissors-like central nervous system.The fruit bat adult is downcut head with dissecting needle after anaesthetizing with CO2, removes mouthpart and compound eye again and can pick out whole brain.Isolated fruit bat larva of a tapeworm or the cercaria of a schistosome central nervous system in three ages is unified behind the brain of adult, put into the culture dish that fills the outer liquid of standard cell lines and shake separation.

The cerebral tissue that is placed on fruit bat in the culture dish is fixed with a U-shaped metal grill IV, capacitor C 1, C2, C3 or C4 in the capacitor bank is in parallel with capacitor C to be regulated oscillator I to make vibrations pointer II is that 5-10Hz, amplitude are 100 μ m vibrations gently organizationally with the frequency by selecting, and regulates oscillator I gradually by narishige III simultaneously and contacts with organizing to guarantee pointer II always.Can see that by microscope many tissue block break away from tissue one after another and are suspended in the solution.After 10 minutes, stop vibrations, place again and begin to carry out the electrophysiology record after making cell and tissue block adherent in 20 minutes.

Three, solution and medication:

Remove Ca ²⁺-Mg ²⁺The ionic group of solion becomes (mg/100mL): 800 NaCl, 20 KCl, 5 NaH ₂PO ₄, 100NaHCO ₃, 100 glucose.The ionic group of liquid becomes (mM) in the electrode: 140 KCl, 0.5 CaCl ₂, 2 MgCl ₂, 5 ethylidene nitrilo tetraacethyl (EGTA) and 5HEPES are with Tris adjusting PH with base to 7.2.Be stored in and be configured to the solution that ultimate density is 2mM at once after-20 ℃ ATP is dissolved in intracellular fluid.The consisting of of the outer liquid of standard (mM): 130 NaCl, 6 KCl, 2.5 CaCl ₂, 5 MgCl ₂, 5 HEPES, 10 glucose.Add 300nM tetraodotoxin (TTX) and 200 μ M CdCl ₂Come the Na and the Ca ionic channel of blocking voltage gate respectively.With the outer liquid of standard is transferred PH to 7.4 with Tris alkali.Potassium channel blocker 4-AP and triethylammonium tetrakis (TEA) and mentioned reagent are all bought from U.S. seat leather agate (Sigma) company.Medication is the administration (Xu et al., 2000) of Y pipe.Delivery tube distance between two tips cell 50-100 μ m, this medication can guarantee thoroughly to change pericellular solution in 20ms.

Four, electrophysiological recording:

Utilize the voltage clamp pattern of full cell record under room temperature (22-25 ℃), the fruit bat axoneuron to be carried out the electrophysiology record.Recording electrode draws to draw on the instrument at two step of the PP-830 type electrode that Japanese Narishige company produces and forms.Electrode resistance in charging electrode during liquid is 10-12M Ω.Under the IX70 type inverted phase contrast microscope that Japanese Olympus (Olympus) produced, the PCS-5200 type piezoelectric ceramic type narishige steering electrode exposing cell of producing with rich thunder (Burleigh) company of the U.S. forms the sealing-in of huge ohm and inhales the broken full cell record that forms.The electric current Axopatch 200B type patch clamp amplifier of being produced by U.S. Ai Kesen (Axon) company of record is that 2KHz gathers with the sample frequency, and the Digidata 1320A type A/D converter of producing by U.S. Axon company is with conversion of signals and import in the computer.The signal processing treatment of Clampfit 8.0 data processing softwares of utilizing U.S. Axon company to produce to being gathered.The command potential of cell is-70mV.

Five, result and analysis:

Fig. 3 amplifies 600 times phase microscope picture with the isolating fruit bat of vibrating method larva of a tapeworm or the cercaria of a schistosome axoneuron in three ages, and 1 among Fig. 3 and 2,3 and 4,5 and 6 represents I, II and III type neurone in the fruit bat larva of a tapeworm or the cercaria of a schistosome central nervous system in three ages respectively.From Fig. 3 as seen, adopt that the isolating fruit bat of the inventive method larva of a tapeworm or the cercaria of a schistosome axoneuron in three ages is in good condition, complete form and A wide selection of colours and designs;

Among Fig. 41,2,3 and 4 is full cell currents of four types that record from isolating fruit bat larva of a tapeworm or the cercaria of a schistosome II in three ages type neurone.From Fig. 4 as seen, the electrophysiologic characteristics with the isolating fruit bat of the inventive method larva of a tapeworm or the cercaria of a schistosome axoneuron in three ages remains intact;

Among Fig. 51 represents isolating fruit bat adult cerebral tissue, wherein O represents the simple eye of fruit bat, this shows that isolated is the forebrain of fruit bat really, represent the position of our mechanical separation in the dashed circle, Fig. 52,3 in represented two phase microscope pictures with 600 times of the isolating fruit bat adult of vibrating method axoneuron amplifications.Therefrom can find out with present method and can isolate fruit bat adult axoneuron, and neuronic state and form remain intact;

Among Fig. 61,2,3 and 4 is full cell currents of four types that record from isolating fruit bat adult neurone.From Fig. 6 as seen, the electrophysiologic characteristics with the inventive method and the isolating fruit bat adult axoneuron of device remains intact.

Therefore the inventive method and device can be used for separating drosophila larvae and adult axoneuron, and neuronic state and form remain intact, and have good electrophysiologic characteristics.

The inventive method also can be widely used in other biological with device and organize single celled the separation, also once utilizes the inventive method and device successfully to separate rat spinal cord neurone, hippocampus of rats and guinea pig myocardium cell etc. as us.

Claims

1, the single celled vibrations tripping device of a kind of biological tissue is characterized in that the upward fixing vibrations pointer (II) that can thereupon shake of earthquake device (I), and oscillator (I) is fixed on the Manipulators (III); Described oscillator (I) is composed in series successively by voltage stabilized source (V), waveform generator (VI), power amplifier (VII) and heat writing recorder (VIII); Wherein waveform generator (VI) is made up of chip IC L8038 and peripheral circuit; The frequency that changes concussion by the resistance that disposes in the peripheral circuit and variable capacity; The waveform that waveform generator (VI) sends links to each other with heat writing recorder (VIII) by a potentiometer W after power amplifier (VII) amplifies, and is used to change the amplitude of concussion.

2, a kind of with the unicellular vibrations separation method of from biological tissue, separating, it is characterized in that: after required isolating biological tissue is fixed, it is that 5-10Hz, amplitude are that 100-200 μ m shakes organizationally with the frequency that the adjusting shaking device makes the vibrations pointer, thereby obtains isolating unicellular.